Indicator platform

ABSTRACT

A novel indicator platform comprises a plurality of 1H-lndol-3-yl indicator compounds that are capable of converting to a signalophore compound in response to an external stimulus. In one class of indicator compounds, the resulting signalophores are 2-benzylideneindoline compounds that are formed by an intermolecular Aldol-type process; in a further class of indicator compounds, the resulting signalophores are 10H-indolo[1,2-a]indole compounds that are formed by an intramolecular Aldol-type process. The indicators can be used in a wide array of applications relating, for example, to biological systems or optical data storage.

PRIORITY CLAIM

This is a U.S. national stage of application No. PCT/EP2010/056212,filed on May 6, 2010. Priority is claimed on the following application:EP, Application No.: 09159639.5, Filed: May 7, 2009, the content ofwhich is incorporated here by reference.

FIELD OF THE INVENTION

The invention discloses of a new class of chromogenic and fluorogenicindicators responding to external stimuli such as biochemicalenvironment, temperature or photo irradiation. The novel indicatorsoffer broad potential in a wide array of applications relating, forexample, to biological systems or optical data storage.

BACKGROUND OF THE INVENTION

Indicators are materials that produce a detectable signal (also denoted“dS”) in response to an external stimulus (also denoted “eS”). Suchstimuli typically include temperature, light (photo-labile orphotochromic indicators), electric field (electrochromic indicators),pressure (piezoelectric indicators), ion concentration (e.g. pHindicators) and biochemical reactivity (e.g. enzyme indicators).

The mechanism of translation of stimulus into detectable signal isillustrated in Scheme I and typically involves the chemical removal ormodification of a labile group (also denoted “LG”) of the indicator in aprocess mediated by the experience of said stimulus.

Removal or modification of the labile group most often yields anactivated signalogen (also denoted “aS”) which typically undergoesfurther transformation which often involves interaction with auxiliaryreagents (also denoted “aR”) to yield a signalophore (also denoted“SP”), the formation of which usually coincides with the occurrence ofsaid detectable signal. Therefore, an indicator represents an activatedsignalogen that is masked, i.e. inactivated by a labile group. Anindicator system (also denoted “IS”) comprises the elements of activatedsignalogen, labile group, auxiliary reagent (optional), detectablesignal and suitable means for interrogation of detectable signal. Theappearance of a detectable signal is a consequence of experiencing anexternal stimulus and hence allows ready detection and/or quantificationof the external stimulus by interrogation with suitable instrumentationor the human eye.

Examples of detectable signal include change in optical density,absorption or emission (e.g. chromogenic, fluorogenic and luminogenicindicators) or change in electric current or potential (electrogenicindicators).

A detectable signal may be transient or persistent in nature: Forexample a fluorogenic enzyme indicator may release a fluorescent entityupon experience of certain enzymatic activity. In this case thedetectable signal is persistent in nature. In contrast a bioluminogenicindicator may emit light in response to the presence of ATP. Theemission of light representing the detectable signal is transient innature.

A detectable signal may be associated with transient or persistentexternal stimuli: For example, pH indicators continuously change colorin response to changing proton concentrations, which represents apersistent external stimulus. In this case the corresponding indicatorsundergo reversible change and are termed reversible indicator systems.In contrast a photo-labile indicator may undergo lasting color change inresponse to a short laser pulse. In this case the correspondingindicators undergo irreversible change upon a single experience ofexternal stimulus and are termed irreversible indicator systems.

There exist a large number of important technical applications includingbiology, diagnostics, chemistry and optical data storage that relate toirreversible indicators.

Indicators used in biology often respond to enzymatic activity. Enzymeindicators are used in many formats of assay. Such assays may beperformed in solution phase which requires a soluble indicator system.

Other assays require the physical location of enzymatic activity. Here,a suitable indicator will stain the specific site associated with enzymeactivity. For this purpose the indicator must form an insolubleprecipitate in response to localized enzymatic stimuli. These types ofindicators are termed insoluble or precipitating indicator systems.Precipitating indicator systems by nature are irreversible.

Detection of enzyme activity is important in diagnostic and testingapplications as well as in biochemistry, molecular biology and histologyresearch. In diagnostics, enzyme activity relates to the presence ofmicrobial pathogens, as well as to metabolic ill functions or to geneticdisorders.

Immunological methods are based on the interaction of antibodies withantigens. In order to detect such interaction, antibodies must becarrying a label. Enzymes are commonly used to label secondaryantibodies. Hence, the detection of enzyme activity is fundamental toimmunological assays. Immunological assays are widely used in clinicaldiagnostics, food and environmental testing, as well as in biochemicalprotocols such as Western blotting.

In molecular biology the detection of enzyme activity is needed inreporter gene protocols. Genetic expression of a reporter gene givesrise to enzyme activity that can be detected. This way, the researcherobtains information on genetic transformations.

1H-Indox-3-yl Indicator Systems

1H-Indox-3-yl (also denoted “Indox”) indicators are well knownchromogenic indicators widely used to visualize enzymatic activity inmicrobiology, immunology, biochemistry and genetics.

Indox indicator systems are derived from the 1H-indol-3-ol(3-hydroxyindol, respectively its tautomeric form indolin-3-one or3-oxoindoline, denoted Indoxol) structure where the hydrogen atom of the3-hydroxyl group is replaced by a labile group. Loss of the labile groupyields Indoxol as an activated signalogen which interacts withatmospheric oxygen (a common auxiliary reagent) in a complex radicalchain reaction to yield colloidal indigo stains as signalophor. Adramatic change in optical transmission of a sample associated withindigo dye formation represents a detectable signal of the indicatorsystem.

Despite their widespread use and commercial significance, applicationsof Indox indicator systems suffer from some major limitations:

For instance, Indox indicator systems depend on molecular oxygen orother oxidizing auxiliary reagents to develop desired indigosignalophores. Due to said requirement indoxyl substrates are of limitedor no use under anaerobic conditions. Considering the portion of enzymeassays that are performed in the absence of oxygen, this limitation issignificant.

Therefore, indicators with properties similar to indoxyl indicatorswithout the undesirable dependence on molecular oxygen or otheroxidizers would constitute a major improvement over the state of theart.

Most popular Indox indicators yield stains ranging from violet to greenbecause red indigos are less effective for the purpose of staining.

However, it would be desirable to have available a choice of colors whenusing chromogenic or fluorogenic indicator systems. This is ofparticular importance in dual or multiple enzyme assays requiringparallel detection or in applications that require optical contrastagainst off white background.

Therefore, novel chromogenic enzyme indicators that expand the currentcolor selection into the range of yellow to red would represent afurther valuable addition to the art.

Moreover, common enzyme indicators are either chromogenic or fluorogenicin nature: For example, common commercial enzyme indicators based onfluorophores such as 7-hydroxycoumarines (soluble fluorogenicindicators) or quinazolines (precipitating fluorogenic indicators—seefor instance: EP 0 641 351 A1) lack significant absorption in thevisible electromagnetic band and thus escape detection by the human eyewithout the application of optical instrumentation for interrogation. Incontrast, common chromogenic indicators such those derived from3-indoxyls lack fluorescence. This is true in particular forprecipitating indicators since fluorescence in the solid state is a rarephenomenon (due to the well known effect of self-quenching of excitedmolecules arranged in a tight lattice).

It would be advantageous over the current state of the art if enzymeindicators were made available that have both chromogenic andfluorogenic properties.

Diazonium Staining

In histology, another type of indicator is well known and often used tolocalize enzyme activity. This important method was pioneered bySeligman et al. (J. Histochem. Cytochem. 1954, 2, 209-229), Burstone etal. (J. Histochem. Cytochem. 1956, 4, 217-226) and Rutenburg et al. (J.Histochem. Cytochem. 1958, 6, 122-129). It is based on the reaction ofstabilized diazonium salts with electron rich aromatic amines andphenols to form azo dyes. Many azo dyes are of intense color and someare fluorescent.

This diazonium coupling reaction will proceed with aromatic amines andphenols much faster than with their corresponding esters and amides.Therefore, hydrolytic enzyme activity can be detected by exposing asample to a suitable phenolic ester or anilide followed by staining withdiazonium salts. Depending on the substrate and the diazonium salts,good localization can be achieved.

In practice, different types of chromogens such as naphtylamines ornaphthol derivatives are employed. There exists considerable variety ofcommercially available substrates of this type along with suitablediazonium staining salts.

Pearson et al. (Proc. Soc. Exptl. Biol. Med. 1961, 108, 619-613; Lab.Invest. 1963, 12, 712-720), Yarborough et al. (J. ReticuloendothelialSoc. 1967, 4, 390-408), Gossrau et al. (Histochemistry, 1987, 397-404)and others developed staining methods that are based on the reaction ofIndox indicators with diazonium electrophiles for use in histology.

Mohler and Blau (Proc. Natl. Acad. Sci. USA 1996, 12423-12427) haveevaluated many indicator systems for beta-D-galactosidase combiningIndox indicators and various commercial diazonium salts.

It should be noted, however, that due to the carcinogenicity and hightoxicity towards humans and cell cultures and the otherwise hazardousnature of diazonium salts this method poses significant danger to theuser and is not compatible with either in vivo or non-destructive typeof assaying in general.

In view of all the above, a superior type of indicator releasingactivated signalogen which spontaneously yield signalophores in aprocess that is entirely independent of external factors such asmolecular oxygen or any reagent or chemical species present in thesurrounding environment would be desirable.

Metal Chelates

Another class of precipitating substrates are based on metal chelatingmolecules that form insoluble complexes with metal ions. Chelatingmolecules contain two or more functional groups that coordinate to ametal ion. These functional groups can be masked by labile groups toprevent the formation of a metal complex which represents a relativelycommon design of indicator.

Enzyme indicators based on metal chelates do have some practicalrelevance. For example, substrates derived from 8-hydroxyquinolines suchas 8-hydroxyquinoline-beta-D-glucuronide or the naturally occurringesculetin (esculin-beta-D-glucopyranoside) can be used to detect thecorresponding enzymes in the presence of iron(III) salts.

However, these indicators suffer from other drawbacks: Many of thesechelating molecules display biocidal effects on culture. Such toxicityis intrinsic to any metal chelating agents since they generallyinterfere with the functioning of metallo-enzymes. Further, one mustmaintain a certain concentration of metal ions present in the assay,which may cause undesirable interference. In addition, most stainsproduced from metal chelation are brown and hence will not provide goodcontrast from background.

For example, 8-hydroxyquinoline is an excellent ligand that forms stablecomplexes with many transition metals. Masking the hydroxyl group with asuitable labile group produces potentially useful indicators. The assaywill release 8-hydroxyquinoline as the activated signalogen whichrapidly binds iron(II or III) thereby forming a precipitating metalcomplex that is dark colored. Moreover, 8-hydroxyquinoline possessessignificant anti-microbial activity which may inhibit the growth of someprominent organisms within the format of a microbial assay. Accordingly,enzyme indicators preferably should not involve chelating agents orunnatural metal ion concentrations which by nature interfere with thecourse of the assay.

SUMMARY OF THE INVENTION

The present invention aims at overcoming severe limitations of currentindicators including:

-   -   The requirement of auxiliary staining reagents such as oxygen,        oxidizers, metal ions, drastic pH conditions or toxic diazonium        salts.    -   The presence of toxic reagents, intermediates or products that        potentially impact the assay.    -   The limitation of indicator color of precipitating chromogenic        indicators to the green-blue-violet range (lacking yellow to        red).    -   The lack of choice in precipitating enzyme indicators that        exhibit fluorogenic properties.    -   The lack of precipitating enzyme indicators that integrate        chromogenic and fluorogenic properties.

The present invention is based on the discovery that 1H-Indox-3-ylcompounds and derivatives thereof efficiently undergo inter- andintramolecular aldol type reactions yielding novel types of dyessuitable for the purpose of indicating events or environmental changes.

The term “aldol condensation” is customarily used for a two stepchemical reaction between an aldol donor group C₁—(C═O)—C₂ or itstautomer C₁—C(OH)═C₂ (i.e. the enol form) and an aldol acceptorC₃—(C═O)—Y. The reaction involves (1) the formal addition of the formerto the carbonyl group of the latter, thereby forming a carbon-carbonbond between C₂ of the donor and the carbonyl carbon atom of theacceptor, and (2) the subsequent elimination of either water (H₂Z, Z═O)or of HY, depending on the nature of the rest Y. Equivalent reactionswherein either one of the two carbonylic oxygens is replaced by aheteroatom containing species Z such as NH or S are termed hetero-aldolcondensations. In the following, the term “aldol” processes will beintended to also include hetero-aldol processes unless specificallynoted otherwise. The general principle of aldol condensations isdepicted in Scheme II.

In the above, if Y represents an effective leaving group (more likely todepart than ZH) such as halogen, cyano, thiocyano, and optionallysubstituted ammonium, hydroxyl, mercapto, and sulfonyl, the formation ofthe condensation product involves the elimination of HY (Scheme II,bottom-right structure). Otherwise, the loss of H₂Z (typically water)leads to the alternative condensation product (Scheme II, bottom-leftstructure).

An introductory overview of intermolecular and intramolecular aldolcondensation routes according to the present invention including acomparison with prior art is presented in Scheme III.

In the above

-   -   R_(a) and R_(b) represent hydrogen or C1-4 alkyl,    -   R₁, R₂, R₃, R₄, R₆, R₇, R₈, R₉ and R₁₀ are independently        selected from the group consisting of hydrogen, C1-4 alkyl; C1-4        alkoxy; fused or linearly connected aryl; fused or linearly        connected heteroaryl; halogen; cyano; nitro; formyl; and        optionally substituted amino, carboxy, carbonyl, hydroxy and        sulfonyl;    -   R₅ represents either hydrogen, R₁₁ or R₁₂,        -   wherein R₁₁ is

-   -   -   and R₁₂ is

-   -   X is selected from O, NH and S, and    -   MDAB denotes 2-methoxy-4-(N,N-dimethylamino)benzaldehyde.

Hereinabove and in the following, the term “optionally substituted”stands for substitution with a general inert moiety, wherein the term“generally inert” refers herein to any moieties that do not generallyinterfere with the procedural practices of this invention.Representative examples of generally inert groups or moieties maygenerally include hydrogen and such organic groups as, for instance,aromatics to include phenyl, alkyl- and/or halogen-substituted phenyl,naphthyl, phenyl-, alkyl- and/or halogen-substituted naphthyl; saturatedorganic residues to include linear and branched alkyl, for example,methyl, ethyl, propyl to include cyclopropyl, butyl to includecyclobutyl and methyl-substituted cyclopropyl, pentyl to include, e.g.,cyclopentyl and methyl-substituted cyclobutyl, hexyl to include, e.g.,cyclohexyl, methyl-substituted cyclopentyl and dimethyl orethyl-substituted cyclobutyl, heptyl to include cycloheptyl, etc., octylto include cyclooctyl, etc.; halogen-substituted alkyl to includehalogen-substituted cycloalkyl, e.g., fluoroalkyl, perfluoroalkyl, e.g.,trifluoromethyl, and chloralkyl; alkoxy, e.g., methoxy, aromatic-oxy,e.g., phenoxy; alkylthioxy, e.g., methylthioxy; aromatic-thioxy, e.g.,phenylthioxy; acyl, e.g., benzoyl and acetyl, and so forth and the like.

According to one aspect of the invention, an indicator system fordetecting an external stimulus, comprises an indicator compound of thegeneral formula

wherein:

-   X is O, NH or S;-   LG is a labile group with the X-LG moiety being susceptible to    conversion by action of said external stimulus;-   R₁, R₂, R₃ and R₄ are independently selected from the group    consisting of hydrogen, C1-4 alkyl; C1-4 alkoxy; fused or linearly    connected aryl; fused or linearly connected heteroaryl; halogen;    cyano; nitro; formyl; and optionally substituted amino, carboxy,    carbonyl, hydroxy and sulfonyl;-   R₅ is either hydrogen or R₁₂, wherein R₁₂ is

wherein:

-   Z is O, NH or S;-   R₆, R₇, R₈, R₉ and R₁₀ are independently selected from the group    consisting of hydrogen, C1-4 alkyl; C1-4 alkoxy; fused or linearly    connected aryl; fused or linearly connected heteroaryl; halogen;    cyano; nitro; formyl; and optionally substituted amino, carboxy,    carbonyl, hydroxy and sulfonyl;    and wherein, if R₅ is hydrogen, the indicator system further    comprises an acceptor compound of the general formula

wherein R_(a) and R_(b) are independently selected from hydrogen andC1-4 alkyl.

According to one embodiment of the indicator system based on anintermolecular aldol reaction, R₅ is hydrogen and the acceptor compound(B) is 2-methoxy-4-(N,N-dimethylamino)benzaldehyde, which has beendetermined preferable for the intended purpose.

Depending on the application of the indicator system, LG will be chosenfrom a large variety of possible labile groups, many of which are knownin the art. In particular, LG is selected from the group consisting ofbeta-D-galactopyranoside, tert-butyldimethylsilyloxy (TBDMS), acetate,choline phosphate, alpha-D-glucopyranoside, beta-D-glucuronide sodiumsalt, N-acetyl-beta-D-galactopyranoside and beta-D-glucopyranoside.

According to one embodiment of the indicator system based on anintramolecular aldol reaction, R₁₀ is selected from the group consistingof hydrogen, methyl, methoxy, phenyl, DMP or heteroaryl including, CFur,Fur, NPyr, wherein:

According to a further aspect of the invention, a method of detecting anexternal stimulus in a region of interest comprises the steps of:

-   -   providing the region of interest with an indicator system as        defined above; and    -   monitoring for a signal from a signalophore species formed as a        consequence of said external stimulus;

The indicator system comprises an indicator compound having a labilegroup LG bound to a heteroatom X, wherein X is O, NH or S, the X-LGmoiety being susceptible to conversion by action of said externalstimulus, said conversion leading to formation of an active signalogenspecies (aS) comprising a moiety wherein X is bound to a carbon atomthat is bound to a further carbon atom by a double bond; and said activesignalogenic species possessing aldol donor properties thereby promotingevents of aldol condensations. Therefore, said active signalogen species(aS) yield signalophore species in the presence of suitable aldolacceptor molecules, said acceptor molecules containing reactivesubstructures selected from the group of carbonyl, imino andthiocarbonyl.

In a preferred embodiment based on an intermolecular aldol condensation,the acceptor moiety is a carbonyl moiety and is provided by employmentof an acceptor compound of the general formula

wherein R_(a), and R_(b) are independently selected from hydrogen andC1-4 alkyl, thereby providing an indicator system wherein thesignalophore species is derived from the general structure of2-benzylideneindoline (C) as shown below.

In this embodiment, the acceptor compound (B) is preferably chosen as2-methoxy-4-(N,N-dimethylamino)benzaldehyde.

In a particularly preferred embodiment based on an intramolecular aldolreaction, the acceptor moiety is a part of said indicator molecule and,if R₅ is R₁₂, the signalophore species is a 10H-indolo[1,2-a]indole withthe structural formula

wherein R₁₃ is either OH or R₁₀.

Note: The numbering of R_(x) is maintained from indicator tosignalophore, but nomenclature changes. Therefore, chemical names ofstructures (D) are derived of the following numbering Scheme:

In one embodiment, the method defined hereinabove is carried out undersubstantially oxygen-depleted conditions.

According to yet another aspect of the invention, there is provided amethod of preparing an indicator compound of the general formula (A′)

wherein:

-   X is O, NH or S;-   LG is a labile group with the X-LG moiety being susceptible to    conversion by action of said external stimulus;-   R₁, R₂, R₃ and R₄ are independently selected from the group    consisting of hydrogen, C1-4 alkyl; fused or linearly connected    aryl; fused or linearly connected heteroaryl; halogen; cyano; nitro;    formyl; and optionally substituted amino, carboxy, carbonyl, hydroxy    and sulfonyl; and R₁₂ is

wherein: Z is O, NH or S;

-   and wherein R₆, R₇, R₈, R₉ and R₁₀ are independently selected from    the group consisting of hydrogen, C1-4 alkyl; fused or linearly    connected aryl; fused or linearly connected heteroaryl; halogen;    cyano; nitro; formyl; and optionally substituted amino, carboxy,    carbonyl, hydroxy and sulfonyl. The method comprises the step of    N-arylation of an indoxyl compound of the general formula

with a benzene derivative of the general formula

wherein Q is a leaving group selected from iodo, bromo, triflate andtosylate, preferably iodo or bromo.

According to a further aspect of the invention, there is provided acompound of structural formula (G)

wherein

-   X is O, NH or S-   R₁, R₂, R₃, R₄, R₆, R₇, R₈, R₉ and R₁₃ are independently selected    from the group consisting of hydrogen, C1-4 alkyl; fused or linearly    connected aryl; fused or linearly connected heteroaryl; halogen;    cyano; nitro; formyl; and optionally substituted amino, carboxy,    carbonyl, hydroxy and sulfonyl. These compounds (G) are generally    classified as indolo[1,2-a]indoles (henceforth also denoted as    “IO”).

A number of compounds of formula (G) have been mentioned in Rogness andLarock (Tetrahedron Letters 2009, 50, 4003-4008), which purportedly wasmade available on 14 Apr. 2009. Accordingly, a preferred embodimentencompasses the above defined compounds of structural formula (G) withthe exception of the compounds listed in Tables 2 and 3 of Rogness andLarock (see claim 14).

According to a further aspect of the invention, a compound of structuralformula (G) is used in an indicator system for detecting an externalstimulus. Such external stimuli include but are not limited to: Heatingor temperature variations; electromagnetic irradiation; applied electricpotentials; particular chemical environments such as acidic, alkaline,oxidizing or reducing; presence of particular chemical species such asions, enzymes, oxygen or oxidizing agents, hydrogen or reducing agents;presence of particular biological species such as viruses, bacteria,fungi, antibodies, cells and cellular organelles, cellular tissue; andeven plants, animals and humans as well as organs, bodily fluids, wasteor decay thereof.

The indicators or indication systems according to the invention areparticularly useful for the purpose of staining cells, microbialcolonies or cell tissue.

According to one embodiment, staining or indication with the IOindicators of this invention is used in combination with classic Indoxstaining or indication.

The manifold uses of IO indicators include but are not limited tostaining of bacterial colonies growing in blood cultures, staining offungal cultures, staining for labelling of individual cells, microbialcolonies or cell tissue, staining for the purpose of inducingfluorescence within cellular tissue, colonies of cells, cells, cellularstructures or organelles.

Moreover, IO staining is useful for the purpose of imaging staticconditions such as size, location, enumeration and spread of benign ormalignant cellular tissue, cells, cellular structures, cell walls,membranes, compartments, organelles, antibodies, chromosomes, genomes,genes, plasmids, vectors, nucleic acid strands, proteins or enzymes.

Furthermore, IO staining is useful for the purpose of visualizingdynamic events such as diffusion, growth or decay of benign or malignantcellular tissue, individual cells, cellular structures, organelles,antibodies, chromosomes, genomes, genes, plasmids, vectors, nucleic acidstrands, proteins or enzymes, and processes of phagocytosis andpathogenesis.

Still further, IO staining is useful for the purpose of recording,retrieving, storing or archiving digital information.

DETAILED DESCRIPTION OF THE INVENTION

The above mentioned and other features and objects of this invention andthe manner of achieving them will become more apparent and thisinvention itself will be better understood by reference to the followingdescription of various embodiments of this invention taken inconjunction with the accompanying drawings, wherein:

FIG. 1 shows UV/visible absorption spectra (in arbitrary absorbanceunits “AU”) of various 10H-indolo[1,2-a]indole stains (labelled “IOx”)and of the corresponding beta-D-galactosidase indicators (labelled“Ixa”) (see Tables Ia and Ic for symbol definitions): a) x=14; b) x=16;c) x=19; d) x=21; e) x=17; f) x=15;

FIG. 2 shows fluorescence emission spectra in relative fluorescenceunits (RFU) for excitation at 360 nm of bacterial colonies stained withvarious 10H-indolo[1,2-a]indoles (see Table Ia for symbol definitions);and

FIG. 3 shows fluorescence excitation spectra in relative fluorescenceunits (RFU) for emission at 550 nm of bacterial colonies stained withvarious 10H-indolo[1,2-a]indoles (see Table Ia for symbol definitions).

INTERMOLECULAR ALDOL INDICATORS: MDAB STAINING AND BI INDICATOR SYSTEMS

In this first section a novel method termed MDAB aldol staining isdisclosed. The method is based on the discovery that certain1H-Indox-3-yl activated signalogens and2-methoxy-4-(N,N-dimethylamino)benzaldehyde (MDAB) auxiliary reagentefficiently undergo an intermolecular aldol condensation to yield darkred to brown colored precipitates suitable for the purpose ofindication, thereby providing a novel indicator system significantlyexpanding upon the current art.

The basis of the MDAB staining technique disclosed here is the discoverythat Indox activated signalogens possess significant aldol donorproperties and that such properties are highly specific towards certainaldol acceptors, in particular MDAB.

Specifically, it was discovered that widely used commercial IndoxIndicators releasing Indoxol activated signalogen5-bromo-4-chloro-1H-indol-3-ol (Table Ib, aS4) and5-bromo-6-chloro-1H-indol-3-ol (Table Ib, aS5) produce dark violetstains derived of the 2-benzylideneindolin-2-one parent structure (TableIc, entries 11-22).

Further, it was recognized that said aldol donor properties of Indoxactivated signalogens can be moderated or eliminated by masking the X—Hmoiety in position 3 by replacing the hydrogen with a labile group.

In principle, Indox indicators currently used in indicator systemsincluding oxygen or other oxidizers as auxiliary reagents can be usedwith MDAB as the auxiliary reagent instead.

However, in practice, not all Indox activated signalogens (or theindicators derived thereof) form efficient aldol donor/acceptor pairswith MDAB: For instance the commonly used 5-bromo-4-chloro-1H-indol-3-ol(Table Ib, aS4) and 5-bromo-6-chloro-1H-indol-3-ol (Table Ib, aS5)activated signalogens proved far more effective than6-chloro-1H-indol-3-ol (Table Ib, aS3).

For example, O-silylated 5-bromo-4-chloro-1H-indol-3-ol (Table Ia, I4b)was used as a simple indicator for fluoride ions (external stimulus):The silyl group is labile towards fluoride ions in the presence of whichthe 5-bromo-4-chloro-1H-indol-3-ol (aS4) is set free to react with MDABto yield mentioned insoluble pink precipitate (Table Ic, BI4).

In a second example, a blend of the commercial reagent5-bromo-4-chloro-1H-indol-3-yl-beta-D-galactopyranoside (Table Ia, I4a)and MDAB yielded said pink dye upon incubation with beta-D-galactosidasein the absence of oxygen while it is well known in the art that I4a byitself depends on the presence of oxygen or an oxidizer for functioningas an indicator.

Further, Indox activated signalogens 3-aminoindole or 3-mercaptoindole(Table Ib, aS6 and aS7) were tested for their aldol donor properties.While 3-aminoindole failed to yield any aldol product with MDAB (TableIc, BI6), 3-mercaptoindole and MDAB did produce the characteristic pinkdye (BI7) under strongly acidic conditions (Tables IIa-c, entries 14 and15).

Indox indicator systems tested are listed in Tables IIa (aerobic, noMDAB), IIb (aerobic, MDAB) and IIc (anaerobic, MDAB).

Further elaboration revealed that Indox/MDAB indication can be used tostain microbial colonies by using characteristic biomarker enzymes asexternal stimulus. Importantly, MDAB showed no toxic effect on microbialcell growth in these studies. Results are summarized in Table III.

For example, microbial plating media containing the I4a/MDAB indicatorsystem were incubated in the presence and absence of atmospheric oxygen:While at atmospheric concentration of oxygen the formation of turquoiseindigo (Table Ic, IN4) dominated, stained colonies appeared pink due tothe formation of BI4 (Table Ic) under micro-anaerobic or anaerobicconditions due to the effect of MDAB staining (Table III, entry 10).

In general, commercially available Indox indicators derived of aS4 oraS5 in combination with 2 to 4 equivalents of MDAB were found to be thepreferable indicator systems for staining of microbial cultures underanaerobic conditions. Indicators I9a, I10a, I11a, and I12a incombination with MDAB represent indicator systems that are effectiveunder aerobic conditions (Table III, entries 26-37): It appears that theformation of indigo from N-arylated Indox activated signalogens isinefficient and that the intramolecular aldol condensation dominatesinstead.

INTRAMOLECULAR ALDOL INDICATORS: IO INDICATOR SYSTEMS

The MDAB staining disclosed above is based on the unique match of analdol donor and a MDAB aldol acceptor pair. In the following, it isdisclosed that a similar effect can be achieved by chemically linkingaldol donor and acceptor.

It is well known in the art that intramolecular ring-closure reactionsincluding intramolecular aldol reactions proceed at high ratesespecially if 5- or 6-membered rings are formed in the process.Therefore, the concept was developed to connect aldol donor and acceptorsuch that the spatial arrangement of donor and acceptor moieties wouldfavor the event of an intramolecular aldol condensation.

In analogy to the above described MDAB staining, untimely occurrence ofthe aldol condensation can be prevented by the masking effect of alabile group conjugated to the aldol donor. A spontaneous aldolcondensation takes place after departure or modification (e.g. chemicalreduction) of the labile group in response to an external stimulus.

The design of an intramolecular aldol indicator requires the aldolacceptor to be attached to the donor by means of a chemical structurelinking the C₃ atom of the aldol acceptor (Scheme II). Traditional INIndox indicators provide a perfect anchor site for the linker: Theindole nitrogen. Said linking may include one or two sequentiallyarranged atoms C₁ and C₂ as shown in Scheme IV. In the former case aformal intramolecular aldol condensation would yield a 5-membered ringwhereas in the latter case it would yield a 6-membered ring in thesignalophore structure.

-   -   C₁, C₂, C₃ represent carbon atoms optionally substituted by        hydrogen; C1-4 alkyl; fused or linearly connected aryl; fused or        linearly connected heteroaryl; halogen; cyano; thio-cyano;        nitro; nitroso; formyl; and optionally substituted amino,        carboxy, carbonyl, hydroxyl, mercapto and sulfonyl    -   R₁, R₂, R₃, R₄ are selected from the group consisting of        hydrogen, C1-4 alkyl; fused or linearly connected aryl; fused or        linearly connected heteroaryl; halogen; cyano; thiocyano; nitro;        nitroso; formyl; and optionally substituted amino, carboxy,        carbonyl, hydroxyl, mercapto and sulfonyl    -   X is selected from O, NH and S.

The aldol acceptor is preferably chosen as an aromatic carbonylcompound, (hence C₁ and C₂ represent members of an aromatic ring) suchthat an intramolecular aldol condensation would result in the formationof an extended conjugated system thereby providing the signalophore withdesirable optical properties.

Due to the kinetic advantage of an intramolecular aldol reaction donorand acceptor molecules do not need to form a matching donor/acceptorpair such as Indox/MDAB.

Of the many synthetic routes explored to achieve the desired linkingbetween donor and acceptor structures, N-arylation was discovered to bethe preferred method.

N-arylation is a well established synthetic method. For example it iswell known in the art that indoles readily react with iodobenzene toproduce N-phenylindoles in high yields.

For instance 3-indolyl-beta-D-galactoside (I1a) was reacted withderivatives of Iodobenzene in DMF in the presence of a copper catalystto yield the corresponding 1-phenyl-3-indolyl-beta-D-galactopyranoside(I8a). This is remarkable since the labile group was chosen to be acarbohydrate and said carbohydrate was used without chemical protection.

In analogy, novel aldol type indicators were obtained in one simple stepand in high yields by N-arylation of the corresponding commercially1H-indox-3-yl indicators under a variety of conditions includingtraditional Ullmann type copper catalysis or newer variants thereof suchas recently published by Taillefer et al. (Efficient Iron/CopperCo-Catalyzed Arylation of Nitrogen Nucleophiles. Angew. Chem., Int. Ed.2007, 46, 934-936).

-   -   R₁, R₂, R₃, R₄, R₆, R₇, R₈, R₉ and R₁₀ are selected from the        group consisting of hydrogen, C1-4 alkyl; fused or linearly        connected aryl; fused or linearly connected heteroaryl; halogen;        cyano; nitro; formyl and optionally substituted amino, carboxy,        carbonyl, hydroxy and sulfonyl    -   X is selected from O, NH and S.

The concept was demonstrated to be of general validity: A significantnumber of commercial Indox indicators were successfully conjugated to arange of aromatic aldol acceptors by means of the above mentionedN-arylation techniques thereby providing simple, efficient and genericentry into a novel family of indicators (Table Ia, entries 15-46).

Novel indicators generally did respond to suitable external stimuli asanticipated (Tables IVa-c). Removal of the labile masking grouptriggered the postulated aldol condensation.

Ketones, (acting as internal acceptors), generally yielded brightlycolored insoluble dyes with colors ranging from yellow, orange, red tobrown. Occasionally, intense green fluorescence of transient nature wasobserved which is characteristic of 3-indoxyl species such as arerepresented by the aS species, which in the postulated process of aldolcondensation would be of transient nature as well.

Esters (acting as internal acceptors), in contrast, producedpersistently green fluorescent dyes (Table Ic: IO10, IO11, IO12) freelysoluble in water above pH 5.

Upon further acidification said dyes formed orange colorednon-fluorescent precipitates from aqueous solutions. This process wasfound to be completely reversed by the addition of base. Evidently, saiddyes are highly acidic in nature and highly fluorescent in deprotonatedform.

Generally, all of these dyes are derived of a parent indolo[1,2-a]indole(Table Ic, entries 23-46). The indolo[1,2-a]indole structure formallyconsists of two indoles sharing the nitrogen and the adjacent pyrrolecarbon. Despite the appeal and simplicity of this structure,indolo[1,2-a]indoles (henceforth denoted “IO”) appear to be novel.

In FIG. 1 absorption spectra of various IO stains are overlaid with theones of the corresponding indicators. The graphs illustrate that IOabsorption between 300 and 400 nm and 400 and 500 nm are both lacking inthe corresponding indicators and therefore provides useful detectablesignal.

For example incubation of the N-benzophenone conjugate of6-chloroindoxyl-beta-D-galactoside (121a) with beta-D-galactosidase for24 h produced a bright yellow precipitate which was collected, washedwith water, dried and characterized as7-chloro-11-phenyl-10H-indolo[1,2-a]indol-10-one (Table Ic, IO21), whichcorresponds to the expected aldol condensation product.

IO staining was tested on plating media inoculated with variousbacterial beta-D-galactosidase positive strains. Colonies ofbeta-D-galactosidase positive strains of E. coli appeared yellow to reddepending on the indicator used while beta-galactosidase negativeSalmonella colonies appeared no different in the presence or absence oftested galactosidase indicators (Tables IVa and IVb).

Further, IO staining was tested on different bacteria producing avariety of different biomarker enzymes (Table IVc).

It should be noted that IO staining and classical indigo (IN) stainingcan be usefully combined, thereby extending the color scheme (TableIVd): For instance indicator I4h was used in combination with I21a in adual plating media assay staining beta-D-glucosidase andbeta-D-galactosidase bacterial species concurrently. While the strainspositive for the former biomarker stained blue and strains positive forthe latter stained yellow, strains positive for both enzymes stainedgreen (mixture of blue and yellow).

It is well known that colonial growth of facultative anaerobic microbialspecies is relatively slow under anaerobic conditions. Due to theprolonged time of incubation, indicator systems used for the purpose tostain anaerobically cultured colonies must provide excellentlocalization (or minimal diffusion). From the experimental data shown inTable IVe it can be concluded that some IO indicator systems areexcellently suited for said purpose. At the same time the dataillustrate the failure of classic IN staining (I4a) and demonstrate thataccumulation of corresponding activated signalogen caused by the absenceof matching auxiliary reagent (e.g. oxygen) partially inhibits microbialgrowth.

Blood cultures represent a common and essential tool in clinicalmicrobiology. Staining of microbial colonies on agar plates is obscuredby the dark color and lack of translucency of Blood Agar plates.Therefore, IO indicator systems have been evaluated for use on BloodAgars (Table IVf). The experiments produced very favorable results: IOsignalophores provided excellent optical contrast and localization.

Staining of fungal cultures is another important area of application forbiological indicator systems. For this purpose, an IO indicator systemhas been devised which is labile towards the action ofD-galactosamidase, a biomarker enzyme for the notorious pathogen Candidaalbicans. Although colonial growth under the culturing conditions chosenwas modest, in the presence of I21g fungal colonies producing a matchingexternal stimulus turned bright yellow and became readilydistinguishable by the human eye (Table IVg).

All of the plating media listed in Tables IVa-f were inspected underillumination at 366 nm before, during and after incubation. Thereby itwas discovered that 10H-indolo[1,2-a]indol-10-ones are fluorescent inthe solid state and that the effect of fluorescence was maintained by IOstained colonies: Generally, at 366 nm plating media (except for BloodAgar plates) containing IO indicator systems provide a bluish backgroundon which unstained colonies appear as faint shadows, whereas IO stainedcolonies stand out in bright colors ranging from yellow-green tored-brown providing excellent visual contrast and high sensitivity.

The blood ingredients of Blood Agar plates effectively quenchfluorescence. Therefore, plates appear black under 366 nm UV light.Interestingly, fluorescence of IO stains dispersed in colonies growingon Blood Agar plates remains undisturbed and provides spectacularillumination of colonies against the black background (Table IVf).

In the following, the discovery that IO stains induced persistentfluorescence within microbial colonies was further elaborated:Fluorescence could be quantified in colonies harvested from platingmedia by using a standard plate reader. Intensity and wavelength of theemission was strongly dependent on the type of indicator system used.Data collected in various emission scans are presented in FIG. 2.

The corresponding samples were also subjected to excitation scansrecording emission at 550 nm (FIG. 3). Interestingly, IO stains werefound to be excitable between 300 and 500 nm, which is an unusuallybroad range. Clearly, the bright shining colors of many IO stains aredue to the effect of excitation (optical brightening) by ambient light.

In an extension of the above, it was also recognized that standardliquid culturing of E. coli in the presence of IO indicator systemsyielding fluorescent signalophores provides a novel method forfluorescence labeling of individual cells: In samples taken from saidculture, individual cells of E. coli became clearly visible under astandard fluorescence microscope.

IO staining, therefore, may provide a simple and economic method forlive cell labeling, which commonly relies on the elaborate applicationof fluorescent antibodies or genetic vectors that encode fluorescentproteins.

Finally, the scope of the invention was further explored by evaluationof structural variations of IO indicator systems which are derived ofthe nitrogen and sulfur analogues of 1-aryl-1H-indol-3-ol activatedsignalogen.

For said purpose, 1-(2-benzoylphenyl)-1H-indol-3-yl ethyl carbamate(I32) was subjected to alkaline and pig liver esterase catalyzedhydrolysis. A yellow precipitate was isolated, which, by means ofcomparison with a reference sample, was identified as11-phenyl-10H-indolo[1,2-a]indol-10-one (IO19) rather than the expected11-phenyl-10H-indolo[1,2-a]indol-10-imine (IO32). A plausibleexplanation is provided by the possible hydrolysis of the imino groupunder the conditions of assay which would transform IO32 to the observedIO19. Independent of the mechanism of action and its practicalrelevance, the experiment demonstrates that in principle the concept canbe extended to include indicator systems where the labile group isattached to a nitrogen atom. The relevance of this finding lies in amanifold of important biological applications which base on thedetection of amino-peptidase activity and the fact that no usefulprecipitating indicator systems are available in the current state ofthe art.

In a second example, the oxidized form of1-(2-acetylphenyl)-1H-indol-3-thiole (aS33),bis[1-(2-acetylphenyl)-1H-indol-3-yl]-disulfane (I33) was subjected toreduction of the S—S bridge by TCEP, a standard reagent used in proteinbiochemistry, thereby releasing aS33. While no detectable signal wasobserved at neutral conditions, the assay solution turned to yellow,thereby clearly indicating the event of disulfide reduction, whenalkaline conditions where used. This example underlines the fact that IOindicator systems represent a generic and highly versatile concept. Italso serves to emphasize that the IO indicator systems are by no meanslimited to detect external stimuli associated with enzymatic activity.The many examples presented herein below are not to be taken as alimitation of this invention but merely reflect the practical field theinventors are engaged in.

EXAMPLES Example 1 Preparation of Indicators I8 to I31 by N-arylation ofI1 to I4 (Table Ia)

Note: Aromatic iodo compounds were either commercially available orprepared by standard Friedel-Crafts acylation with ortho-iodobenzoicacid. 2-(2-Iodobenzoyl)-N-methylpyrrole was prepared in analogy toCarson et al. (WO2000048584), 2-(2-iodobenzoyl)furan and2-(2-iodobenzoyl)-5-carboxyfuran in analogy to Garland et al.(DE2557956) and 4-chloro-2-iodobenzophenone according to Gabbutt et al.(Tetrahedron 2006, 62, 737-745). 1-Acetyl-2-iodo-4-methoxybenzene and1-benzoyl-2-iodo-4-methoxybenzene were obtained by Friedel-Craftsreaction of 3-iodoanisol in the presence of aluminium chloride.

Experiments were carried out in a Synthos 3000 multimode microwavereactor from Anton Paar GmbH. The following parameters were adjusted:P(max)=1400 W; T(IRmax)=200° C.; Drive: rotation; Stirrer: 3; p-rate=2.0bar/s. Ramp-time=2 min (130° C.) and hold-time=180 min (130° C.). APTFE-liner (100 ml) fitted with a Teflon-coated stirring bar was used asthe reaction vessel. In a typical procedure a mixture of 5 mMol of1H-Indol-3-yl indicator, 10 mMol of the corresponding aryl iodide, 5mMol potassium carbonate, 0.5 mMol copper(I) iodide in 20 ml of DMF wassubjected to MW irradiation. The product mixture was filtered and thesolids washed with 10 ml ethanol. The filtrate was evaporated to drynessand the crude product purified by flash chromatography [e.g. silica gel,toluene/ethanol (5:1)].

IR spectra were recorded as neat solids on a Perkin-Elmer FT-IRspectrophotometer, model Spectrum ONE (v [cm⁻¹]). ¹H- and ¹³C-NMRspectra were recorded at 298 K on Brucker AVANCE-400 [400.13 MHz (¹H),100.61 MHz (¹³C)] spectrometer (δ [ppm], J [Hz]).

1-Phenyl-1H-indol-3-yl-beta-D-galactopyranoside (I8a)

Yield: 628 mg (34%); TLC [toluene/ethanol (5:3)]: R_(f) 0.47.

¹H-NMR [DMSO-d₆]: 7.73-7.71 (m, 1H); 7.56-7.54 (m, 5H); 7.42 (s, 1H);7.37-7.32 (m, 1H); 7.23-7.19 (m, 1H); 7.14-7.10 (m, 1H); 5.30-5.28 (d,1H); 4.89-4.87 (d, 1H); 4.74-4.72 (d, 1H, J=7.8 Hz); 4.71-4.69 (d, 1H);4.54-4.53 (d, 1H); 3.72-3.66 (m, 1H); 3.61-3.56 (m, 3H); 3.48-3.41 (m,2H).

¹³C-NMR [DMSO-d₆]: 139.18, 139.01, 132.28, 129.73, 125.73, 123.33,122.99, 121.42, 119.60, 118.19, 113.35, 110.17, 104.22, 75.71, 73.33,70.39, 68.23, 60.54.

6-Chloro-1-phenyl-1H-indol-3-yl-beta-D-galactopyranoside (I9a)

Yield: 560 mg (28%); TLC [toluene/ethanol (5:3)]: R_(f) 0.43.

¹H-NMR [DMSO-d₆]: 7.72-7.70 (m, 1H); 7.58-7.56 (m, 4H); 7.52-7.51 (m,1H); 7.45 (s, 1H); 7.41-7.37 (m, 1H); 7.16-7.13 (m, 1H); 5.30-5.29 (d,1H); 4.89-4.87 (d, 1H); 4.72-4.71 (d, J=7.8 Hz, 1H); 4.69-4.67 (m, 1H);4.54-4.53 (d, 1H); 3.71-3.63 (m, 2H); 3.59-3.55 (m, 3H); 3.43-3.39 (m,1H).

¹³C-NMR [DMSO-d₆]: 138.75, 138.55, 132.48, 129.85, 127.82, 126.29,123.56, 120.11, 119.99, 119.65, 114.43, 109.81, 104.18, 75.73, 73.23,70.30, 68.18, 60.50.

1-[2-(Methoxycarbonyl)phenyl]-1H-indol-3-yl-beta-D-galactopyranoside(I10a)

Yield: 496 mg (23%); TLC [toluene/ethanol (5:3)]: R_(f) 0.41.

¹H-NMR [DMSO-d₆]: 7.89-7.87 (m, 1H); 7.77-7.69 (m, 2H); 7.56-7.52 (m,2H); 7.25-7.02 (m, 4H); 5.27-5.26 (br. d, 1H); 4.87 (br. s, 1H);4.67-4.65 (d, J=7.8 Hz, 1H); 4.65-4.64 (br. d, 1H); 4.53-4.52 (br. d,1H); 3.71-3.63 (m, 2H); 3.58-3.49 (m, 3H); 3.44-3.39 (m, 1H).

¹³C-NMR [DMSO-d₆]: 166.38, 138.94, 137.68, 133.77, 133.16, 130.73,128.21, 128.04, 127.41, 122.79, 120.96, 119.41, 118.16, 114.62, 109.39,104.54, 75.65, 73.34, 70.50, 68.16, 60.43, 52.24.

6-Chloro-1-[2-(methoxycarbonyl)phenyl]-1H-indol-3-yl-beta-D-galactopyranoside(I11a)

Yield: 526 mg (23%); TLC [toluene/ethanol (5:3)]: R_(f) 0.41.

¹H-NMR [DMSO-d₆]: 7.91-7.89 (m, 1H); 7.78-7.74 (m, 1H); 7.70-7.68 (m,1H); 7.60-7.55 (m, 2H); 7.19 (s, 1H); 7.10-7.08 (m, 1H); 6.98 (d, 1H);4.65-4.63 (d, J=7.8 Hz, 1H); 3.66-3.61 (m, 2H); 3.56-3.51 (m, 3H); 3.48(s, 3H); 3.42-3.39 (m, 1H).

¹³C-NMR [DMSO-d₆]: 165.94, 138.58, 136.99, 134.01, 133.29, 130.77,128.25, 128.14, 127.94, 127.52, 119.68, 119.62, 119.58, 115.73, 109.12,104.46, 75.64, 73.19, 70.35, 68.05, 60.34, 52.23.

5-Bromo-4-chloro-1-[2-(methoxycarbonyl)phenyl]-1H-indol-3-yl-beta-D-galactopyranoside(I12a)

Yield: 572 mg (21%); TLC [toluene/ethanol (5:3)]: R_(f) 0.41.

¹H-NMR [DMSO-d₆]: 7.93-7.91 (m, 1H); 7.81-7.74 (m, 1H); 7.62-7.58 (m,1H); 7.54-7.52 (m, 1H); 7.37-7.34 (m, 1H); 7.27 (s, 1H); 6.87-6.84 (m,1H); 4.70-4.68 (d, J=7.8 Hz, 1H); 3.70-3.63 (m, 2H); 3.54-3.52 (m, 3H);3.49 (s, 3H); 3.43-3.39 (m, 1H).

¹³C-NMR [DMSO-d₆]: 165.56, 139.53, 137.85, 136.76, 133.66, 133.40,130.88, 128.63, 128.38, 128.20, 126.60, 123.67, 116.21, 112.73, 110.12,103.91, 75.59, 73.46, 70.31, 68.05, 60.30, 52.33.

1-(2-Formylphenyl)-1H-indol-3-yl-beta-D-galactopyranoside (I13a)

Yield: 443 mg (22%); TLC [toluene/ethanol (5:3)]: R_(f) 0.42.

¹H-NMR [DMSO-d₆]: 9.64 (s, CHO); 8.01-7.99 (m, 1H); 7.89-7.85 (m, 1H);7.75-7.73 (m, 1H); 7.66-7.59 (m, 2H); 7.45 (s, 1H); 7.26-7.09 (m, 3H);5.29-5.27 (br. d, 1H); 4.89-4.88 (br. d, 1H); 4.75-4.73 (d, J=7.8 Hz,1H); 4.63-4.61 (br. d, 1H); 4.54-4.53 (br. d, 1H); 3.71-3.65 (m, 2H);3.54 (br. s, 3H); 3.43-3.36 (m, 1H).

¹³C-NMR [DMSO-d₆]: 189.40, 141.07, 139.27, 135.46, 135.02, 131.03,128.37, 127.99, 127.91, 123.37, 120.92, 119.88, 118.24, 115.60, 109.66,104.13, 75.61, 73.26, 70.36, 68.09, 60.35.

1-(2-Acetylphenyl)-1H-indol-3-yl-beta-D-galactopyranoside (I14a)

Yield: 1.20 g (60%); TLC [toluene/ethanol (5:3)]: R_(f) 0.38.

¹H-NMR [DMSO-d₆]: 7.75-7.70 (m, 3H); 7.59-7.54 (m, 2H); 7.21 (s, 1H);7.18-7.09 (m, 2H); 7.05-7.03 (m, 1H); 5.29-5.27 (br. d, 1H); 4.87-4.85(br. d, 1H); 4.69-4.67 (d, J=7.8 Hz, 1H); 4.63-4.61 (br. s, 1H);4.52-4.51 (br. d, 1H); 3.71-3.62 (m, 2H); 3.58-3.50 (m, 3H); 3.46-3.39(m, 1H); 1.82 (s, 3H).

¹³C-NMR [DMSO-d₆]: 200.12, 139.13, 137.09, 136.59, 133.75, 132.61,129.14, 127.86, 127.69, 123.10, 120.72, 119.64, 118.16, 114.58, 109.56,104.24, 75.56, 73.23, 70.38, 68.05, 60.28, 28.28.

1-(2-Acetylphenyl)-4-chloro-1H-indol-3-yl-beta-D-galactopyranoside(I15a)

Yield: 1.09 g (49%); TLC [toluene/ethanol (5:3)]: R_(f) 0.41.

¹H-NMR [DMSO-d₆]: 7.79-7.72 (m, 2H); 7.62-7.58 (m, 1H); 7.54-7.53 (m,1H); 7.29 (s, 1H); 7.12-7.07 (m, 2H); 6.97-6.92 (m, 1H); 5.02-5.01 (br.d, 1H); 4.86-4.85 (br. d, 1H); 4.77-4.75 (d, J=7.8 Hz, 1H); 4.63 (br. s,1H); 4.55-4.54 (br. d, 1H); 3.72-3.65 (m, 2H); 3.55 (br. s, 3H);3.46-3.40 (m, 1H); 1.92 (s, 3H).

¹³C-NMR [DMSO-d₆]: 199.75, 138.48, 137.14, 136.00, 135.01, 132.72,129.27, 128.24, 128.07, 124.21, 123.63, 120.41, 117.24, 114.95, 108.65,103.51, 75.53, 73.54, 70.39, 68.07, 60.27, 28.40.

1-(2-Acetylphenyl)-6-chloro-1H-indol-3-yl-beta-D-galactopyranoside(I16a)

Yield: 0.96 g (44%); TLC [toluene/ethanol (5:3)]: R_(f) 0.46.

¹H-NMR [DMSO-d₆]: 7.78-7.70 (m, 3H); 7.62-7.57 (m, 2H); 7.25 (s, 1H);7.17-7.12 (m, 1H); 7.05-7.04 (m, 1H); 5.30-5.29 (br. d, 1H); 4.88-4.86(br. d, 1H); 4.68-4.66 (d, J=7.7 Hz. 1H); 4.63-4.61 (br. s, 1H);4.52-4.51 (br. d, 1H); 3.70-3.62 (m, 2H); 3.56-3.50 (m, 3H); 3.42-3.39(m, 1H); 1.91 (s, 3H).

¹³C-NMR [DMSO-d₆]: 199.95, 138.83, 137.06, 135.83, 133.97, 132.71,129.29, 128.18, 127.85, 125.23, 120.04, 119.70, 119.49, 115.73, 109.38,104.25, 75.62, 73.17, 70.33, 68.04, 60.29, 28.45.

1-(2-Acetylphenyl)-5-bromo-4-chloro-1H-indol-3-yl-beta-D-galactopyranoside(I17a)

Yield: 0.61 g (23%); TLC [toluene/ethanol (5:3)]: R_(f) 0.42.

¹H-NMR [DMSO-d₆]: 7.83-7.80 (m, 1H); 7.77-7.73 (m, 1H); 7.64-7.60 (m,1H); 7.56-7.53 (m, 1H); 7.41-7.39 (m, 1H); 7.35 (s, 1H); 6.92-6.90 (m,1H); 5.07-5.06 (br. d, 1H); 4.87-4.86 (br. d, 1H); 4.74-4.72 (d, J=7.7Hz, 1H); 4.62 (br. s, 1H); 4.55-4.53 (br. d, 1H); 3.71-3.64 (m, 1H);3.55 (br. s, 3H); 3.47-3.39 (m, 2H).

¹³C-NMR [DMSO-d₆]: 199.56, 138.04, 137.03, 135.44, 133.60, 132.77,129.38, 128.54, 128.33, 126.96, 123.77, 118.55, 116.27, 113.05, 110.39,103.72, 75.57, 73.44, 70.32, 68.06, 60.27, 28.50.

1-(2-Acetyl-5-methoxyphenyl)-6-chloro-1H-indol-3-yl-beta-D-galactopyranoside(I18a)

Yield: 1.6 g (65%); TLC [toluene/ethanol (4:1)]: R_(f) 0.36.

FT-IR: 3459m, 2964w, 2927w, 2867w, 1665s, 1601s, 1569m, 1497m, 1471s,1451s, 1372s, 1267s, 1220s, 1140s, 1081s, 1053s, 1020s, 975m, 945m,880m, 800m, 735m, 696m.

¹H-NMR [DMSO-d₆]: 7.80 (d, J=8.7, 1 arom. H); 7.71 (d, J=8.5, 1 arom.H); 7.28 (s, 1 arom. H); 7.17-7.11 (m, 2 arom. H); 7.07 (d, J=2.5, 1arom. H); 7.06 (d, J=1.7, 1 arom. H); 5.30 (d, J=5.0, OH); 4.87 (d,J=5.8, OH); 4.68 (d, J=7.8, H—C(1)_(Gal)); 4.64 (t, J=5.2, OH); 4.52 (d,J=4.7, OH); 3.88 (s, OCH₃); 3.70 (t, J=4.0, H—C(4)_(Gal)); 3.65 (m_(c),1H—C_(Gal)); 3.58-3.51 (m, 3H—C_(Gal)); 3.48-3.39 (m, 1H—C_(Gal)); 1.86(s, CH₃).

¹³C-NMR [DMSO-d₆]: 197.86 (s, C═O); 162.54 (s, C—OCH₃); 138.79, 138.18,134.01 (3 s); 131.68 (d); 129.00 (s); 128.82, 128.12 (2 d); 127.85 (s);125.23, 120.01, 119.66 (3 d); 119.53 (s); 115.67, 113.92, 113.27, 109.44(4 d); 104.22 (d, C(1)_(Gal)); 75.68, 73.22, 70.36, 68.11 (4 d,C(2-5)_(Gal)); 60.38 (t, C(6)_(Gal)); 55.90 (q, OCH₃); 28.03 (q,CH₃—C═O).

1-(2-Benzoylphenyl)-1H-indol-3-yl-beta-D-galactopyranoside (I19a)

Yield: 1.09 g (46%); TLC [toluene/ethanol (5:3)]: R_(f) 0.41.

¹H-NMR [DMSO-d₆]: 7.82-7.81 (m, 1H); 7.80-7.79 (m, 1H); 7.78-7.77 (m,2H); 7.70-7.69 (m, 1H); 7.68-7.67 (m, 3H); 7.64-7.13 (m, 4H); 7.03 (s,1H); 6.99-6.95 (m, 1H); 5.23-5.22 (br. d, 1H); 4.85-4.83 (br. d, 1H);4.68-4.65 (br. s, 1H); 4.51-4.50 (br. d, 1H); 4.29-4.27 (d, J=7.7 Hz,1H); 3.72-3.70 (m, 1H); 3.63-3.52 (m, 3H); 3.42-3.32 (m, 2H).

¹³C-NMR [DMSO-d₆]: 195.54, 138.83, 136.97, 135.68, 135.48, 133.41,132.92, 132.21, 129.92, 128.30, 128.14, 127.86, 127.48, 127.18, 125.24,122.63, 120.61, 119.38, 117.80, 115.27, 109.87, 104.49, 75.40, 73.19,70.31, 67.91, 60.17.

1-(2-Benzoylphenyl)-4-chloro-1H-indol-3-yl-beta-D-galactopyranoside(I20a)

Yield: 0.87 g (34%); TLC [toluene/ethanol (5:3)]: R_(f) 0.52.

¹H-NMR [DMSO-d₆]: 7.82-7.78 (m, 1H); 7.70-7.60 (m, 3H); 7.45-7.41 (m,3H); 7.26-7.22 (m, 2H); 7.10 (s, 1H); 7.05-6.94 (m, 3H); 4.89-4.87 (d,1H); 4.83-4.81 (d, 1H); 4.66-4.64 (br. s, 1H); 4.53-4.52 (d, J=4.7 Hz,1H); 4.43-4.41 (br. s, 1H); 3.72-3.70 (m, 1H); 3.61-3.51 (m, 3H);3.48-3.41 (m, 2H).

¹³C-NMR [DMSO-d₆]: 195.25, 138.20, 136.41, 135.87, 135.58, 134.70,133.14, 132.23, 129.84, 128.48, 128.02, 127.62, 123.76, 123.15, 120.19,117.01, 115.40, 108.94, 103.68, 75.36, 73.46, 70.26, 67.91, 60.10.

1-(2-Benzoylphenyl)-6-chloro-1H-indol-3-yl-beta-D-galactopyranoside(I21a)

Yield: 1.30 g (51%); TLC [toluene/ethanol (5:3)]: R_(f) 0.21.

¹H-NMR [DMSO-d₆]: 7.83-7.79 (m, 1H); 7.71-7.64 (m, 3H); 7.48-7.46 (m,1H); 7.41-7.37 (m, 3H); 7.26-7.13 (m, 3H); 7.07 (s, 1H); 7.01-6.98 (m,1H); 5.24-5.23 (br. d, 1H); 4.86-4.84 (br. d, 1H); 4.68-4.65 (br. s,1H); 4.52-4.50 (br. d, 1H); 4.29-4.27 (d, J=7.5 Hz, 1H); 3.71-3.69 (m,1H); 3.59-3.53 (m, 3H); 3.44-3.33 (m, 2H).

¹³C-NMR [DMSO-d₆]: 195.45, 138.60, 136.32, 135.66, 135.63, 133.66,133.04, 132.35, 129.93, 128.82, 128.41, 128.12, 127.96, 127.49, 127.34,125.23, 119.78, 119.35, 119.28, 116.30, 109.69, 104.49, 75.46, 73.13,70.26, 67.91, 60.18.

1-(2-Benzoylphenyl)-6-chloro-1H-indol-3-yl-alpha-D-glucopyranoside(I21e)

Yield: 1.57 g (61%); TLC [toluene/ethanol (5:3)]: R_(f) 0.28.

¹H-NMR [DMSO-d₆]: 7.83-7.79 (m, 1H); 7.72-7.62 (m, 3H); 7.46-7.44 (m,1H); 7.39 (s, 1H); 7.38-7.35 (m, 2H); 7.21-7.17 (m, 3H); 7.12 (m, 1H);7.00-6.98 (m, 1H); 5.08-5.06 (d, 1H); 5.00-4.99 (d, 1H); 4.96-4.95 (d,1H); 4.91-4.90 (d, J=2.2 Hz, 1H); 4.59-4.57 (m, 1H); 3.68-3.58 (m, 2H);3.54-3.31 (m, 3H); 3.21-3.15 (m, 1H).

¹³C-NMR [DMSO-d₆]: 195.53, 137.91, 136.42, 135.63, 135.45, 133.59,133.00, 132.45, 130.06, 128.34, 127.97, 127.88, 127.56, 127.32, 119.70,119.36, 119.20, 115.82, 109.70, 100.63, 73.53, 73.00, 71.70, 70.01,60.83.

1-(2-Benzoylphenyl)-6-chloro-1H-indol-3-yl-beta-D-glucuronide sodiumsalt (I21f)

Yield: 1.79 g (69%); TLC [toluene/ethanol (5:3)]: R_(f) 0.07.

¹H-NMR [DMSO-d₆]: 7.82-7.79 (m, 1H); 7.69-7.63 (m, 3H); 7.57-7.54 (m,1H); 7.40-7.38 (m, 3H); 7.24-7.20 (m, 2H); 7.11 (m, 2H); 6.99-6.97 (m,1H); 6.66 (br. s, 1H); 5.45 (br. s, 1H); 5.21 (br. s, 1H); 4.30 (br. s,1H); 3.47-3.40 (m, 1H); 3.27-3.22 (m, 3H).

¹³C-NMR [DMSO-d₆]: 195.39, 172.84, 138.69, 136.30, 135.61, 133.73,133.09, 132.41, 129.94, 128.43, 128.02, 127.45, 127.40, 119.79, 119.62,119.38, 118.31, 116.62, 116.57, 109.64, 103.96, 76.43, 73.93, 73.04,71.92.

1-(2-Benzoylphenyl)-6-chloro-1H-indol-3-yl-N-acetyl-beta-D-galactosaminide(I21g)

Yield: 1.46 g (53%); TLC [toluene/ethanol (5:3)]: R_(f) 0.28.

¹H-NMR [DMSO-d₆]: 7.82-7.74 (m, 2H); 7.70-7.64 (m, 2H); 7.62 (d, NH);7.43-7.35 (m, 3H); 7.24-7.17 (m, 3H); 7.09 (d, 1H); 7.02 (s, 1H); 6.98(dd, 1H); 4.73 (d, 1H); 4.63 (t, 1H); 4.60 (d, 1H); 4.42 (d, 1H); 3.93(q, 1H); 3.70 (t, 1H); 3.60-3.48 (m, 3H); 3.40-3.32 (m, 1H); 1.83 (s,3H).

¹³C-NMR [DMSO-d₆]: 195.5, 169.6, 162.3, 138.7, 136.34, 135.72, 133.70,133.15, 132.45, 129.95, 128.5, 128.0, 127.55, 127.5, 120.0, 119.3,118.8, 116.2, 109.8, 103.0, 75.55, 70.8, 67.3, 60.25, 52.0, 23.2.

1-(2-Benzoylphenyl)-5-bromo-4-chloro-1H-indol-3-yl-beta-D-galactopyranoside(I22a)

Yield: 1.14 g (39%); TLC [toluene/ethanol (5:3)]: R_(f) 0.48.

¹H-NMR [DMSO-d₆]: 7.83-7.79 (m, 1H); 7.71-7.61 (m, 3H); 7.46-7.42 (m,3H); 7.34-7.32 (m, 1H); 7.28-7.24 (m, 2H); 7.16 (s, 1H); 7.00-6.98 (m,1H); 4.96-4.95 (br. d, 1H); 4.84-4.82 (br. d, 1H); 4.66-4.65 (br. s,1H); 4.53-4.52 (d, J=4.7 Hz, 1H); 4.38-4.35 (br. s, 1H); 3.72-3.70 (m,1H); 3.61-3.54 (m, 3H); 3.48-3.43 (m, 2H).

¹³C-NMR [DMSO-d₆]: 195.12, 137.81, 135.92, 135.55, 133.32, 133.23,132.30, 129.88, 128.51, 128.35, 128.08, 127.69, 126.55, 123.38, 118.34,116.70, 112.84, 110.64, 103.89, 75.41, 73.37, 70.18, 67.90, 60.10,55.98.

1-(2-Benzoyl-5-chlorophenyl)-6-chloro-1H-indol-3-yl-beta-D-galactopyranoside(I23a)

Yield: 1.28 g (47%); TLC [toluene/ethanol (5:3)]: R_(f) 0.49.

¹H-NMR [DMSO-d₆]: 7.79 (s, 1H); 7.72 (m, 2H); 7.45-7.39 (m, 4H);7.22-7.17 (m, 3H); 7.12 (s, 1H); 7.02-6.99 (m, 1H); 5.23-5.22 (d, 1H);4.85-4.84 (d, 1H); 4.68-4.65 (m, 1H); 4.51-4.50 (d, 1H); 4.29 (br. s,1H); 3.71-3.69 (m, 1H); 3.57-3.52 (m, 3H); 3.44-3.33 (m, 2H).

¹³C-NMR [DMSO-d₆]: 194.51, 138.89, 137.74, 136.51, 135.38, 134.28,133.64, 133.15, 131.65, 128.41, 128.05, 127.93, 127.66, 127.32, 120.05,119.49, 119.24, 116.06, 109.84, 104.36, 75.49, 73.12, 70.23, 67.90,60.19.

1-(2-Benzoyl-5-methoxyphenyl)-6-chloro-1H-indol-3-yl-beta-D-galactopyranoside(I24a)

Yield: 2.4 g (76%); TLC [toluene/ethanol (4:1)]: R_(f) 0.23.

FT-IR: 3367m, 2911w, 2853w, 1654s, 1600s, 1579m, 1499m, 1470m, 1448m,1365m, 1264s, 1239s, 1118s, 1075s, 1022s, 975m, 923m, 872s, 750m, 703s.

¹H-NMR [DMSO-d₆]: 7.66 (d, J=8.6, 1 arom. H); 7.44 (d, J=8.5, 1 arom.H); 6.36-7.32 (m, 1 arom. H); 7.34 (s, 1 arom. H); 7.32 (s, 1 arom. H);7.21-7.14 (m, 6 arom. H); 6.98 (dd, J=8.5, 1.8, 1 arom. H); 5.23 (d,J=4.9, OH); 4.85 (d, J=5.7, OH); 4.69 (t, J=5.5, OH); 4.51 (d, J=4.7,OH); 4.29 (br. s, H—C(1)_(Gal)); 3.92 (s, OCH₃); 3.71 (t, J=4.0,H—C(4)_(Gal)); 3.60-3.53 (m, 3H—C_(Gal)); 3.46 (t, J=6.2, 1H—C_(Gal));3.39-3.34 (m, 1H—C_(Gal)).

¹³C-NMR [DMSO-d₆]: 194.80 (s, C═O); 162.32 (s, C—OCH₃); 138.58, 138.49,136.35, 133.58 (4 s); 132.46, 132.14, 128.16, 127.67 (4 d); 127.48,127.44 (2 s); 119.73 (d); 119.46 (s); 119.21, 116.35, 113.68, 112.35,109.75 (5 d); 104.42 (d, C(1)_(Gal)); 75.56, 73.17, 70.28, 68.00 (4 d,C(2-5)_(Gal)); 60.32 (t, C(6)_(Gal)); 55.87 (q, OCH₃).

1-[2-(2,4-Dimethoxybenzoyl)phenyl]-1H-indol-3-yl-beta-D-galactopyranoside(I25a)

Yield: 1.28 g (48%); TLC [toluene/ethanol (5:3)]: R_(f) 0.45.

¹H-NMR [DMSO-d₆]: 7.70-7.65 (m, 1H); 7.55-7.47 (m, 4H); 7.09-6.95 (m,5H); 6.26-6.23 (m, 2H); 5.25-5.23 (d, 1H); 4.86-4.84 (d, 1H); 4.69-4.66(br. s, 1H); 4.51-4.50 (d, 1H); 4.33-4.31 (d, J=7.7 Hz, 1H); 3.69 (s,3H); 3.61-3.54 (m, 4H); 3.44 (s, 3H); 3.48-3.41 (m, 1H); 3.39-3.34 (m,1H).

¹³C-NMR [DMSO-d₆]: 193.05, 163.77, 159.66, 138.91, 138.70, 136.42,133.78, 131.89, 131.33, 128.95, 127.21, 126.78, 122.30, 120.45, 119.50,119.02, 117.73, 115.10, 109.65, 105.23, 104.75, 97.55, 75.50, 73.18,70.35, 68.01, 60.32, 55.41, 55.38.

1-[2-(2,4-Dimethoxybenzoyl)phenyl]-1H-indol-3-yl choline phosphate(I25d)

Yield: 0.92 g (68%); TLC [ethyl acetate/pyridine/acetic acid/water(30:25:5:15)]: R_(f) 0.46.

¹H-NMR [DMSO-d₆]: 7.68-7.62 (m, 1H); 7.48 (m, 2H); 7.43 (2 d, 2H); 7.20(d, 1H); 7.10 (s, 1H); 7.05 (t, 1H); 7.02 (t, 1H); 6.94 (t, 1H); 6.35(d, 1H); 6.32 (dd, 1H); 4.05 (m, 2H); 3.72 (s, 3H); 3.53 (s, 3H); 3.46(t, ³J[H,P]=4.6 Hz, 2H); 3.03 (s, 9H).

¹³C-NMR [DMSO-d₆]: 193.0, 164.1, 160.0, 157.0, 138.8, 136.7, 134.0 (d,J[C,P]=7.4 Hz), 133.6, 132.3, 131.3, 129.0, 127.0 (J[C,P]=2.4 Hz),122.0, 121.6 (J[C,P]=6.3 Hz), 119.5, 118.9, 117.6, 116.1, 109.7, 105.7,97.9, 65.4 (J[C,P]=4.8 Hz), 59.0 (J[C,P]=5.36 Hz), 55.5 (2 OCH₃), 53.0.

4-Chloro-1-[2-(2,4-dimethoxybenzoyl)phenyl]-1H-indol-3-yl-beta-D-galactopyranoside(I26a)

Yield: 1.76 g (62%); TLC [toluene/ethanol (5:3)]: R_(f) 0.54.

¹H-NMR [DMSO-d₆]: 7.70-7.66 (m, 1H); 7.58-7.53 (m, 2H); 7.49-7.47 (m,1H); 7.12-7.10 (m, 1H); 7.02 (s, 1H); 7.01-6.99 (m, 1H); 6.96-6.93 (m,2H); 6.34-6.29 (m, 2H); 4.85-4.83 (br. s, 2H); 4.67-4.64 (br. s, 1H);4.53-4.52 (d, 1H); 4.48-4.46 (d, J=5.9 Hz, 1H); 3.73 (s, 3H); 3.72-3.70(m, 1H); 3.64-3.55 (m, 3H); 3.48 (s, 3H); 3.46-3.38 (m, 2H).

¹³C-NMR [DMSO-d₆]: 192.84, 163.99, 159.72, 139.25, 138.17, 135.77,135.07, 131.92, 131.33, 128.91, 127.18, 123.74, 122.88, 119.84, 119.36,116.86, 115.14, 108.77, 105.40, 103.94, 97.59, 75.50, 73.44, 70.32,68.00, 60.24, 55.48, 55.40.

6-Chloro-1-[2-(2,4-dimethoxybenzoyl)phenyl]-1H-indol-3-yl-beta-D-galactopyranoside(I27a)

Yield: 1.88 g (66%); TLC [toluene/ethanol (5:3)]: R_(f) 0.45.

¹H-NMR [DMSO-d₆]: 7.72-7.67 (m, 1H); 7.57-7.56 (m, 2H); 7.52-7.50 (m,2H); 7.12-7.10 (m, 1H); 7.02 (s, 1H); 7.00-6.98 (m, 2H); 6.31-6.26 (m,2H); 5.27-5.26 (d, 1H); 4.87-4.86 (d, 1H); 4.69-4.66 (br. s, 1H);4.52-4.51 (d, J=4.6 Hz, 1H); 4.38-4.35 (br. s, 1H); 3.71 (s, 3H);3.70-3.68 (m, 1H); 3.60-3.55 (m, 3H); 3.48 (s, 3H); 3.48-3.43 (m, 2H).

13C-NMR [DMSO-d6]: 192.89, 163.89, 159.54, 138.89, 138.52, 135.69,133.98, 131.92, 131.54, 129.06, 127.77, 127.21, 127.03, 119.36, 119.21,119.18, 116.09, 109.42, 105.38, 104.68, 97.41, 75.59, 73.13, 70.30,68.03, 60.36, 55.44, 55.32.

5-Bromo-4-chloro-1-[2-(2,4-dimethoxybenzoyl)phenyl]-1H-indol-3-yl-beta-D-galactopyranoside(I28a)

Yield: 2.17 g (67%); TLC [toluene/ethanol (5:3)]: R_(f) 0.58.

¹H-NMR [DMSO-d₆]: 7.72-7.67 (m, 1H); 7.61-7.49 (m, 3H); 7.34-7.31 (m,1H); 7.12-7.10 (m, 1H); 7.06 (s, 1H); 6.88-6.86 (m, 1H); 6.34-6.31 (m,1H); 6.28-6.27 (m, 1H); 4.95-4.94 (d, 1H); 4.85-4.84 (d, 1H); 4.67-4.64(br. s, 1H); 4.53-4.52 (d, J=4.6 Hz, 1H); 4.45 (br. s, 1H); 3.73 (s,3H); 3.72-3.70 (m, 1H); 3.64-3.51 (m, 3H); 3.47 (s, 3H); 3.46-3.37 (m,2H).

¹³C-NMR [DMSO-d₆]: 192.76, 163.98, 159.63, 139.30, 137.84, 135.27,133.68, 131.82, 131.42, 128.97, 128.14, 127.24, 126.32, 123.33, 119.28,118.17, 116.38, 112.51, 110.45, 105.40, 104.06, 97.48, 75.53, 73.36,70.24, 67.99, 60.24, 55.48, 55.39.

1-[2-(5-Carboxylfuranoyl)phenyl]-6-chloro-1H-indol-3-yl-beta-D-galactopyranoside(I29a)

Yield: 0.99 g (36%); TLC [toluene/ethanol (5:3)]: R_(f) 0.

¹H-NMR [CD₃COOD]: 7.82-7.74 (m, 2H); 7.69-7.67 (m, 1H); 7.63-7.59 (m,1H); 7.55-7.49 (m, 1H); 7.33-7.32 (m, 1H); 7.12 (s, 1H); 7.04-7.02 (m,1H); 6.97-6.96 (d, 1H); 6.88-6.87 (d, 1H); 4.69-4.67 (d, J=7.2 Hz, 1H);4.14-4.13 (d, 1H); 4.04-3.94 (m, 3H); 3.86-3.82 (m, 2H).

¹³C-NMR [CD₃COOD]: 184.9, 154.0, 147.7, 139.6, 138.2, 135.2, 135.1,133.6, 130.9, 129.8, 128.4, 127.8, 121.3, 120.6, 119.8, 119.5, 119.1,116.6, 110.9, 104.3, 75.5, 73.9, 71.9, 69.8, 62.1.

6-Chloro-1-(2-furanoylphenyl)-1H-indol-3-yl-beta-D-galactopyranoside(I30a)

Yield: 1.65 g (66%); TLC [toluene/ethanol (5:3)]: R_(f) 0.38.

¹H-NMR [DMSO-d₆]: 7.84 (m, 1H), 7.80-7.76 (m, 1H); 7.72-7.70 (m, 1H);7.64-7.57 (m, 3H); 7.18 (s, 1H); 7.10-7.03 (m, 3H); 6.54-6.53 (m, 1H);5.28-5.27 (d, 1H); 4.87-4.85 (d, 1H); 4.66-4.63 (m, 1H); 4.52-4.51 (d,1H); 4.47-4.45 (d, J=7.7 Hz, 1H); 3.71-3.69 (m, 1H); 3.63-3.53 (m, 3H);3.48-3.41 (m, 2H).

¹³C-NMR [DMSO-d₆]: 181.55, 151.01, 148.81, 138.58, 136.06, 135.24,133.78, 132.32, 129.62, 127.83, 127.62, 127.56, 120.83, 119.85, 119.48,119.42, 116.24, 112.61, 109.64, 104.50, 75.54, 73.16, 70.32, 68.00,60.27.

6-Chloro-1-[2-(N-methylpyrrole-2-carbonyl)phenyl]-1H-indol-3-yl-beta-D-galactopyranoside(I31a)

Yield: 1.76 g (69%); TLC [toluene/ethanol (5:3)]: R_(f) 0.36.

¹H-NMR [DMSO-d₆]: 7.95 (m, 1H); 7.73-7.69 (m, 1H); 7.63-7.55 (m, 3H);7.17 (m, 1H); 7.10 (s, 1H); 7.05-7.02 (m, 2H); 6.35-6.34 (m, 1H);5.91-5.90 (m, 1H); 5.27-5.26 (d, 1H); 4.86-4.84 (d, 1H); 4.63-4.60 (br.s, 1H); 4.51-4.50 (d, 1H); 4.45-4.43 (d, J=7.7 Hz, 1H); 3.71-3.68 (m,1H); 3.61-3.33 (m, 5H); 2.88 (s, 3H).

¹³C-NMR [DMSO-d₆]: 183.78, 162.23, 138.49, 137.27, 135.67, 133.72,132.73, 131.16, 129.86, 129.41, 127.44, 127.34, 127.18, 121.99, 119.62,119.34, 116.07, 109.87, 108.05, 104.62, 75.42, 73.14, 70.29, 67.86,60.08, 36.40.

Example 2 Preparation of I19c and I25c by N-arylation of I1c (Table Ia)

5.0 mMol aryliodide, 1.31 g (7.48 mMol) I1c and 0.67 g (5.47 mMol)copper(I) acetate in 25 ml dry dimethylacetamide were heated to 150° C.for two hours under a nitrogen atmosphere. The product mixture wascooled to room temperature and diluted with 25 ml ethyl acetate. Thesuspension was poured onto 50 ml saturated aqueous ammoniumchloridesolution and stirred for 30 minutes. The organic phase was washed withbrine, dried with sodium sulfate, filtered and evaporated to dryness.The resulting oil was purified by column chromatography on silica gel[toluene; toluene/acetone (10:1)].

1-(2-Benzoylphenyl)-1H-indol-3-yl acetate (I19c)

Yield: 590 mg (33%); TLC (toluene): R_(f) 0.15.

¹H-NMR [DMSO-d₆]: 7.83-7.79 (m, 1H); 7.71-7.64 (m, 3H); 7.42-7.36 (m,3H); 7.32-7.30 (m, 1H); 7.26-7.25 (m, 1H); 7.23-7.11 (m, 4H); 7.05-7.01(m, 1H); 2.27 (s, 3H).

¹³C-NMR [DMSO-d₆]: 195.27, 168.03, 136.36, 135.78, 135.63, 133.41,133.08, 132.23, 130.69, 129.91, 128.49, 128.11, 127.94, 127.61, 122.89,120.24, 120.04, 118.37, 117.35, 110.27, 20.46.

1-[2-(2,4-Dimethoxybenzoyl)phenyl]-1H-indol-3-yl acetate (I25c)

Yield: 1.23 g (59%); TLC [toluene/acetone (10:1)]: R_(f) 0.66.

¹H-NMR [DMSO-d₆]: 7.71-7.67 (m, 1H); 7.56-7.52 (m, 3H); 7.37-7.35 (m,1H); 7.31 (s, 1H); 7.18-7.16 (m, 1H); 7.14-7.10 (m, 3H); 7.06-7.02 (m,1H); 6.30 (s, 1H); 3.70 (s, 3H); 3.49 (s, 3H); 2.30 (s, 3H).

¹³C-NMR [DMSO-d₆]: 192.77, 167.99, 163.99, 139.07, 135.72, 133.60,131.92, 131.31, 130.42, 128.92, 128.79, 128.09, 127.65, 127.13, 125.21,122.64, 119.75, 118.24, 117.16, 110.09, 105.52, 97.58, 55.38, 55.33,20.51.

Example 3 Preparation of I32 by N-arylation of I6 (Table Ia)1-(2-Benzoylphenyl)-1H-indol-3-yl ethyl carbamate (I32)

1.30 g (4.22 mMol) 2-iodobenzophenone, 1.29 g (6.32 mMol)3-aminoindol-3-ethylcarbamate and 0.57 g (4.65 mMol) copper(I) acetatein 25 ml dry dimethylacetamide were heated to 150° C. for two hoursunder a nitrogen atmosphere. The product mixture was cooled to roomtemperature and diluted with 25 ml ethyl acetate. The suspension waspoured onto 50 ml saturated aqueous ammoniumchloride solution andstirred for 30 minutes. The organic phase was washed with brine, driedwith sodium sulfate, filtered and evaporated to dryness. The resultingoil was purified by column chromatography on silica gel [toluene;toluene/acetone (10:1)].

Yield: 100 mg (6%); TLC [toluene]: R_(f) 0.10.

¹H-NMR [DMSO-d₆]: 9.50 (s, NH); 7.81-7.77 (m, 1H); 7.71-7.60 (m, 4H);7.41-7.36 (m, 4H); 7.20-7.16 (m, 3H); 7.11-7.07 (m, 1H); 6.99-6.95 (m,1H); 4.13 (q, 2H); 1.24 (t, 3H).

¹³C-NMR [DMSO-d₆]: 195.42, 153.57, 136.98, 135.73, 135.43, 133.92,132.95, 132.15, 129.89, 128.42, 127.86, 127.43, 127.13, 122.59, 121.06,119.21, 118.44, 117.53, 117.08, 109.78, 60.15, 14.53.

Example 4 Preparation of bis[1-(2-acetylphenyl)-1H-indol-3-yl]-disulfane(I33, Table Ia) 1-(2-Acetylphenyl)-1H-indol-3-yl-isothiuronium iodide

1.80 g (7.65 mMol) 1-(2-acetylphenyl)-1H-indole (prepared by the methodof Example 1) and 1.17 g thiourea (15.4 mMol) were dissolved in amixture of 35 ml methanol (35 ml) and 4 ml water. 3.90 g iodine (15.4mMol) was dissolved in a solution of potassium iodide (3.82 g, 23 mMol)in water (7.7 ml). This iodine solution was added dropwise to thesolution above at ambient temperature during an hour and warmed to 35°C. for three hours. Solvents were removed by evaporation. The residualoil was dissolved in ethyl acetate (40 ml). The solution was extractedthree times with water (3×10 ml). The organic layer was dried andtreated with charcoal (1 g). The crude product was obtained afterevaporation of the filtrate and purified by column chromatography onsilica gel (100 g, ethyl acetate).

Yield: 1.27 g (38%).

¹H-NMR [DMSO-d₆]: 9.06 (s, NH₂); 8.62 (s, NH₂); 8.18 (s, 1H); 7.98 (d,1H); 7.84 (t, 1H); 7.70 (2 t, 2×1H); 7.61 (m, 1H); 7.30 (m, 2H); 7.12(m, 1H); 2.18 (s, 3H).

¹³C-NMR [DMSO-d₆]: 199.4, 170.2, 139.3, 137.8, 136.2, 135.0, 133.1,129.9, 129.3, 129.0, 128.5, 123.9, 122.0, 118.2, 111.0, 92.6, 29.0

Bis[1-(2-acetylphenyl)-1H-indol-3-yl]-disulfane (I33)

1-(2-Acetylphenyl)-1H-indol-3-yl-isothiuronium iodide (0.47 g, 1.07mMol) was dissolved in a mixture of degassed dioxane (2.7 ml) anddegassed water (1.57 ml) under a nitrogen atmosphere.

A degassed 5% aqueous solution of sodium hydroxide (0.8 ml, 1.22 mMol)was added dropwise at ambient temperature, whereby the solution turneddark-brown. The solution was stirred for 48 hours at 45° C. The productmixture was evaporated, dissolved in water (5 ml) and extracted withethyl acetate. The organic layers were dried over sodium sulfate andevaporated leaving the crude product as viscous oil which was purifiedby silica gel flash chromatography.

Yield: 70 mg (24%).

¹H-NMR [DMSO-d₆]: 7.84 (dd, 1H); 7.66 (dt, 1H); 7.62 (dt, 1H); 7.60 (d,1H); 7.55 (s, 1H); 7.38 (d, 1H); 7.19 (2 t, 2×1H); 7.03 (d, 1H); 1.94(s, 3H).

¹³C-NMR [DMSO-d₆]: 199.1, 137.5, 136.4, 135.3, 132.8, 129.6, 128.8,128.7, 128.5, 123.5, 121.3, 119.3, 110.4, 108.0, 28.4.

Example 5 Preparation of 10H-indolo[1,2-a]indoles (Table Ic)

Enzymatic reactions were performed in 100 mM Na₂PO₄ buffer, pH 7.3containing 1 mM MgCl₂ and 670 mg/l enzyme substrate. E. colibeta-galactosidase or K. lactis lactase were added to a finalconcentration of 1000 U/I or 1500 U/I, respectively. 30 ml reactionvolumes were incubated for 5-18 hours at 37° C. with gentle shaking. Thereaction product was collected by centrifugation (4400 g, 15 min),pellets were washed with 10 ml H₂O and centrifuged again (4400 g, 15min).

11-Hydroxy-10H-indolo[1,2-a]indol-10-one (IO10)

¹H-NMR [DMSO-d₆]: 7.73 (d, J=8.2 Hz, 2 arom. H); 7.69 (d, J=7.7 Hz, 2arom. H); 7.50 (t, J=7.3 Hz, 2 arom. H); 7.07 (t, J=7.5 Hz, 2 arom. H);5.79 (br. s).

¹³C-NMR [DMSO-d₆]: 160.75 (s, C═O); 138.32 (s, arom. C); 132.10 (d,arom. C—H); 127.15 (s, arom. C); 123.27, 121.52 (2 d, arom. C—H); 118.65(s, C(10a); 111.72 (d, arom. C—H).

Note: IO10 turns to be readily water-soluble above a pH of 5.

10H-Indolo[1,2-a]indol-10-one (IO13)

¹H-NMR [DMSO-d₆]: 7.94 (m, 1H); 7.84 (m, 1H); 7.74 (m, 1H); 7.67 (m,1H); 7.62 (m, 1H); 7.48 (m, 1H); 7.35 (m, 1H); 7.18 (m, 1H); 7.17 (m,1H).

¹³C-NMR [DMSO-d₆]: 180.7, 144.8, 136.3, 135.0, 133.7, 132.0, 128.5,128.4, 124.9, 124.7, 124.3, 122.2, 112.2, 112.0, 108.1.

MS (CI): 220.2 ([M+H]⁺).

11-Methyl-10H-indolo[1,2-a]indol-10-one (IO14)

¹H-NMR [DMSO-d₆]: 7.85 (m, 1H); 7.77 (m, 1H); 7.71 (m, 1H); 7.62 (m,1H); 7.59 (m, 1H); 7.47 (m, 1H); 7.16 (m, 1H); 7.13 (m, 1H); 2.50 (s,3H).

¹³C-NMR [DMSO-d₆]: 180.8, 144.0, 135.8, 133.3, 132.6, 131.7, 128.6,128.5, 124.1, 123.4, 122.8, 121.5, 121.3, 111.7, 111.6, 8.8.

UV: λ_(max)=255, 355, 435 nm.

9-Chloro-11-methyl-10H-indolo[1,2-a]indol-10-one (IO15)

¹H-NMR [DMSO-d₆]: 7.88-7.86 (m, 1H); 7.76-7.72 (m, 2H); 7.60-7.56 (m,1H); 7.51-7.47 (m, 1H); 7.21-7.17 (m, 1H); 7.12-7.10 (m, 1H); 2.49 (s,3H).

¹³C-NMR [DMSO-d₆]: 178.3, 145.6, 136.8, 133.2, 133.0, 132.3, 130.8,128.8, 124.8, 124.3, 123.0, 122.2, 121.9, 112.0, 110.8, 8.9.

UV: λ_(max)=245, 365, 440 nm.

7-Chloro-11-methyl-10H-indolo[1,2-a]indol-10-one (IO16)

¹H-NMR [DMSO-d₆]: 8.00-7.98 (m, 1H); 7.92 (m, 1H); 7.73-7.71 (m, 1H);7.58-7.56 (m, 1H); 7.50-7.46 (m, 1H); 7.20-7.15 (m, 2H); 2.48 (s, 3H).

¹³C-NMR [DMSO-d₆]: 179.9, 144.8, 140.4, 133.4, 132.9, 132.1, 128.9,127.7, 125.6, 123.5, 123.0, 122.6, 122.0, 112.24, 112.21, 9.00.

UV: λ_(max)=245, 360, 430 nm.

8-Bromo-9-chloro-11-methyl-10H-indolo[1,2-a]indol-10-one (IO17)

¹H-NMR [DMSO-d₆]: 7.96-7.94 (m, 1H); 7.90-7.87 (m, 1H); 7.76-7.72 (m,2H); 7.53-7.48 (m, 1H); 7.22-7.18 (m, 1H); 2.50 (s, 3H).

¹³C-NMR [DMSO-d₆]: 177.3, 144.6, 139.5, 133.2, 132.9, 131.9, 130.7,129.2, 126.0, 123.2, 123.1, 122.2, 116.9, 112.2, 112.1, 9.0.

UV: λ_(max)=245, 365, 450 nm.

7-Chloro-3-methoxy-11-methyl-10H-indolo[1,2-a]indol-10-one (IO18)

¹H-NMR [DMSO-d₆]: 8.07 (d, J=1.6, 1 H); 7.61 (d, J=8.8, 1 H); 7.56 (d,J=8.0, 1H); 7.39 (d, J=2.1, 1 H); 7.17 (dd, J=8.0, 1.6, 1 H); 6.80 (dd,J=8.8, 2.2, 1 H); 3.94 (s, OCH₃); 2.46 (s, CH₃).

11-Phenyl-10H-indolo[1,2-a]indol-10-one (IO19)

¹H-NMR [DMSO-d₆]: 8.04-8.02 (m, 1H); 7.99-7.92 (m, 4H); 7.71-7.65 (m,2H); 7.59-7.55 (m, 3H); 7.51-7.47 (m, 1H); 7.29-7.19 (m, 2H).

¹³C-NMR [DMSO-d₆]: 180.1, 144.0, 136.0, 133.9, 130.7, 130.2, 128.9,128.8, 124.6, 124.5, 124.3, 123.7, 122.7, 112.4, 112.3.

UV: λ_(max)=245, 370, 460 nm.

9-Chloro-11-phenyl-10H-indolo[1,2-a]indol-10-one (IO20)

¹H-NMR [DMSO-d₆]: 8.05-8.03 (m, 1H); 7.97-7.90 (m, 4H); 7.67-7.63 (m,1H); 7.59-7.55 (m, 3H); 7.51-7.49 (m, 1H); 7.31-7.27 (m, 1H); 7.20-7.18(m, 1H).

¹³C-NMR [DMSO-d₆]: 145.5, 137.0, 133.7, 131.0, 130.5, 129.0, 128.8,125.4, 124.9, 124.4, 123.8, 123.1, 112.6, 111.1.

8-Bromo-9-chloro-11-phenyl-10H-indolo[1,2-a]indol-10-one (IO22)

¹H-NMR [DMSO-d₆]: 8.04-8.00 (m, 2H); 7.98-7.92 (m, 3H); 7.89-7.86 (m,1H); 7.60-7.48 (m, 4H); 7.31-7.28 (m, 1H).

¹³C-NMR [DMSO-d₆]: 144.3, 139.5, 133.7, 131.0, 130.4, 130.2, 129.0,128.9, 128.7, 126.2, 125.4, 123.9, 123.1, 117.5, 112.6, 112.5.

7-Chloro-3-methoxy-11-phenyl-10H-indolo[1,2-a]indol-10-one (IO24)

¹H-NMR [DMSO-d₆]: 8.13 (d, J=1.6, 1 H); 7.94, 7.92 (2 br. s, 2H); 7.77(d, J=9.0, 1 H); 7.59 (d, J=8.0, 1 H); 7.54 (br. t, J=8.0, 2 H);7.48-7.44 (m, 2H); 7.21 (dd, J=8.0, 1.6, 1 H); 6.87 (dd, J=9.0, 2.2, 1H); 3.37 (s, OCH₃).

11-(5-Carboxyfuran-2-yl)-7-chloro-10H-indolo[1,2-a]indol-10-one (IO29)

¹H-NMR [DMSO-d₆]: 8.57-8.54 (m, 1H); 8.08-8.06 (m, 1H); 8.00 (m, 1H);7.84 (m, 1H); 7.71-7.69 (m, 1H); 7.61-7.57 (m, 1H); 7.36-7.33 (m, 1H);7.26-7.24 (m, 1H); 6.85-6.84 (m, 1H).

7-Chloro-11-(furan-2-yl)-10H-indolo[1,2-a]indol-10-one (IO30)

¹H-NMR [DMSO-d₆]: 8.33-8.30 (m, 1H); 8.16-8.14 (m, 1H); 8.09 (m, 1H);8.03 (m, 1H); 7.86-7.83 (m, 1H); 7.70-7.68 (m, 1H); 7.60-7.56 (m, 1H);7.34-7.31 (m, 1H); 7.27-7.25 (m, 1H); 6.82-6.81 (m, 1H).

¹³C-NMR [DMSO-d₆]: 177.7, 148.0, 145.0, 144.2, 140.2, 133.9, 129.4,128.3, 127.4, 125.8, 125.2, 124.0, 123.1, 114.0, 113.2, 112.7, 112.6,112.4.

Conclusion: IO stains are produced efficiently from correspondingindicators subjected to enzymatic external stimuli (eS).

Example 6 Optical Absorption of 10H-indolo[1,2-a]indoles (FIG. 1)

Dried 10H-Indolo[1,2-a]indole pellets obtained from procedures ofexample 5 were dissolved in ethanol/DMF (1:1) at 2.5 mM. This stocksolution was diluted to 0.22 mM in 100% EtOH and a spectral scan wasrecorded on a Spectramax M5 (Molecular Devices). The spectra of thecorresponding indicators were recorded for comparison. Data are shown inFIG. 1.

Absorption spectra of Indicators and corresponding IO stains aresubstantially different in the visible band.

Conclusion: IO staining produces strong and readily detectable signals.

Example 7 In-vitro Indication of Fluoride and Hydroxide Ions withIndicator Systems I4b/MDAB and I4c/MDAB

I4b (0.4 g, 1.11 mMol) and MDAB (0.25 g, 1.39 mMol) were dissolved inethanol (8 ml). The resulting indicator solution was kept under anatmosphere of nitrogen gas. A 1 mM solution of tetrabutylammoniumfluoride in THF (1.6 ml) was added to the indicator solution at roomtemperature. After 10 minutes BI4 precipitated as intensely violet heavysolid which was collected and dried (0.19 g, 42%). Similarly, BI4 wasobtained by exposing I4c/MDAB to aqueous base.

¹H-NMR [DMSO-d₆]: 9.84 (s, H1); 7.68 (d, J=8.6 Hz, H6); 7.61 (d, J=8.8Hz, H6′); 7.07 (s, ═CH—); 7.02 (d, J=8.6 Hz, H7); 6.38 (dd, J=8.8 Hz,H5′); 6.24 (d, H3′); 3.88 (s, OCH₃); 3.02 (s, N(CH₃)₂).

¹³C-NMR [DMSO-d₆]: 181.0 (C═O), 160.0, 152.8, 152.5, 138.3, 130.74,130.71, 129.9, 118.3, 113.0, 111.4, 110.0, 109.1, 104.9, 94.6 (OCH₃),55.5 (N(CH₃)₂).

Note: There are two possible stereoisomers of BI4,(E)-5-bromo-4-chloro-2-(4-dimethylamino-2-methoxybenzylidene)indolin-3-oneand(Z)-5-bromo-4-chloro-2-(4-dimethylamino-2-methoxybenzylidene)indolin-3-one.NOE NMR measurements indicate that only (Z)-BI4 was formed in theprocess.

Conclusion: (1) indicator system I4b/MDAB is potentially useful for theanaerobic detection of fluoride ions and (2) indicator system I4c/MDABis potentially useful for the irreversible detection of transientalkaline pH conditions (common pH indicators allow reversible detectiononly).

Example 8 Reaction of MDAB with indol-3-amine (aS6) and indol-3-thiole(aS7)

Exposure of active signalogen aS6 (prepared by reduction of thecorresponding nitroso compound) to atmospheric oxygen yielded apolymeric brown precipitate rather than the bis-imino-indigo derivative.In the presence of MDAB and in the absence of oxygen aS6 produced yellowcolor probably the Schiff base which, however, escaped isolation.Formation of the expected hetero aldol product was not observed.

Exposure of active signalogen aS7 (obtained in analogy to I33) to oxygenyields the corresponding disulfane rather than the bis-thion-indigoanalogue. In the presence of MDAB and in the absence of oxygen theexpected violet color appeared only under strongly acidic conditions.

Conclusion: MDAB staining may not be suitable for indicators derived ofindol-3-amines or indol-3-thiols.

Example 9a MDAB Staining of Bacterial Colonies with Indicators I4a andI5a (Table III, Entries 1-25)

Tryptic Soy Agar (TSA) with yeast extract (0.6%) was used as the baseplating medium. The media were autoclaved at 121° C. for 15 minutes andplaced in a 50° C. water bath to cool. Indicators were added to thebasal medium at a final concentration of 40 mg/100 ml (from 80 mg/mlstock solutions in DMF). Over night cultures of enteric bacterial cellsin Brain Heart Infusion Broth were streaked onto the plates andincubated at 35° C. for 24 hours under aerobic and anaerobic conditions.After incubation, the colonial morphologies on the plates were recorded(Table III).

Under anaerobic conditions and in the absence of MDAB indicators testedfailed to stain bacterial colonies producing suitable external stimulus(beta-D-galactosidase). Weak staining was observed occasionally due toresidual oxygen (micro-anaerobic conditions). Under aerobic conditionsindigo (IN-stains) generally dominated the effects of MDAB staining. Forexample indicator system I4a/MDAB stained colonies violet underanaerobic but indigo blue under aerobic conditions.

Conclusion: MDAB staining is a potentially valuable method for detectionand isolation of microbial species especially under anaerobicconditions.

Note: 4-(N,N-Dimethyl)aminobenzaldehyde (DAB) and a large number ofother potentially suitable Adol acceptors selected from the group ofaromatic carbonyl compounds did not produce any BI-stains under allconditions tested when used to replace MDAB.

Example 9b MDAB Staining of Bacterial Colonies with Indicators I9a,I10a, I11a and I12a (Table III, Entries 26-37)

Indicators were tested with live cultures on Nutrient Agar plates.Nutrient Agar (5 g/l peptone, 5 g/l NaCl, 2 g/l yeast extract, 1 g/lbeef extract, 13 g/l agar, pH 7.4) was autoclaved and allowed to cool to50° C. Then substrates were added to final concentrations of 150 mg/l(from a 20 mg/ml stock solution in DMF). Optionally MDAB was added to aconcentration of 1 mM (from a 200 mM stock solution in DMF) and plateswere poured. Agar plates were inoculated with cultures of Escherichacoli (NM1) or Salmonella enteritidis (RKI 05/07992) (pre-grown onnutrient broth for 8-18 hours) and aerobically incubated at 37° C.Results were recorded after 48 h of incubation and are shown in TableIII.

Conclusion: Indicators I9a, I11a and I12a are potentially useful forMDAB staining under aerobic conditions.

Example 10 10H-Indolo[1,2-a]indole (10) Staining of Bacterial Colonies(Table IVa-c)

Nutrient Agar (5 g/l peptone, 5 g/l NaCl, 2 g/l yeast extract, 1 g/lbeef extract, 13 g/l agar, pH 7.4) was autoclaved and allowed to cool to50° C. Then indicators were added to final concentrations of 150 mg/l(from a 20 mg/ml stock solution in DMF).

Optionally IPTG was added to 100 mg/l (from a 100 mg/ml stock solutionin H₂O) and plates were poured. Agar plates were inoculated withcultures of beta-galactosidase positive Eschericha coli (NM1) orbeta-galactosidase negative Salmonella enteritidis (RKI 05/07992)(pre-grown on nutrient broth for 8-18 hours) and incubated at 37° C.Plates were inspected at 16, 20, 24 and 48 hours and pictures wererecorded at 18, 24, and 48 hours with a Digistore 2 Image documentationsystem (CAMAG) under white light. Data are shown in Tables IVa, IVb andIVc.

A considerable number of indicators molecules (Table IVa, IVb) and aselection of labile groups (Table IVc) were tested with variousmicrobial species producing different biomarker enzymes (externalstimuli, eS). The color of stains ranges from yellow to red. Some stainstested show excellent localization of external stimuli (enzyme activity)while others were significantly soluble in the medium and may be of usefor solution based assay (Table IVa). Staining is accompanied byfluorescence while green fluorescence was observed to be of transientnature, (caused by fluorescence of the active Signalogen)10H-indolo[1,2-a]indoles fluorescence at longer wavelength (yellow-red)was stable.

Conclusion: IO staining is a potentially valuable method for detectionand isolation of microbial species providing a new color scheme, longwave fluorescence and complete independence of auxiliary reagents (aR)potentially interfering with the assay.

Example 11 Indigo (IN) and 10H-indolo[1,2-a]indole (10) ConcurrentStaining (Table IVd)

Nutrient Agar containing 150 mg/l1-(2-benzoylphenyl)-6-chloro-1H-indol-3-yl-beta-D-galactopyranoside(I21a), 100 mg/l IPTG and 75 mg/l5-bromo-4-chloro-1H-indol-3-yl-beta-D-glucopyranoside (I4h) wasinoculated with Escherichia coli, Klebsiella pneumoniae, Enterococcusfaecalis and Enterobacter aerogenes. Data collected after 24 h ofincubation at 37° C. are given in Table IVd. Microbial coloniesproducing both beta-D-galactosidase and beta-D-glucosidase appearedgreen due to parallel formation of blue IN and the yellow IO stains.

Conclusion: The combination of complementary IN and IO staining ispotentially useful to extend the currently available color scheme.

Example 12 IN and IO Staining of Bacterial Colonies under AnaerobicConditions (Table IVe)

Nutrient Agar plates containing 150 mg/l of various substrates and 100mg/l IPTG were inoculated with Escherichia coli. Plates were put in ananaerobic jar (Anaerojar, Oxoid), an AnaeroGen sachet (Oxoid) was added,the jar was closed and incubated for 24 hours at 37° C. Data wererecorded and are shown in Table IVe.

IN staining cannot be used under anaerobic conditions. While due toslower colonial growth diffusion of some IO stains increases underanaerobic conditions other stains such as IO22 produced from indicatorI22a provide perfect localization.

Conclusion: IO staining is a potentially valuable technique for useunder micro-aerobic or anaerobic conditions.

Example 13 IO Staining of Bacterial Colonies on Blood Agar Plates (TableIVf)

Standard commercially available Blood Agar plates were impregnated with150 mg/l1-(2-benzoylphenyl)-6-chloro-1H-indol-3-yl-beta-D-galactopyranoside(I21a) and 100 mg/l IPTG. The plate was inoculated with Klebsiellapneumoniae. Results after incubation at 37° C. for 24 hours are given inTable IVf.

IO stain contrasts very well with the agar plate. Further, despite thequenching effect of blood IO fluorescence is fully maintained andreadily detectable.

Conclusion: IO staining is potentially valuable for use in microbialblood culture.

Example 14 IO Staining of Fungal Colonies (Table IVg)

Agar base (6 g/l soytone, 1.5 g/l yeast extract, 1 g/l glucose, 10 mMsodium phosphate buffer, 13 g/l agar, pH 7.0) was autoclaved and allowedto cool to 50° C. Then 1 mM MnSO₄, 1 g/l N-Acetyl-D-galactosamine and:1-(2-benzoylphenyl)-6-chloro-1H-indol-3-yl-N-acetyl-beta-D-galactosaminide(I21g) were added to final concentrations of 0.64 mM (from a 50 mM stocksolution in DMF) and plates poured. Plates were inoculated with Candidaalbicans, Candida krusei and Saccharomyces cerevisiae and incubated at37° C. for 48 hours. Results were recorded and are shown in Table IVg.

Example 15 Fluorescence of 10H-indolo[1,2-a]indoles Stained MicrobialCells (FIG. 2)

Plating media from Example 10 were examined under 366 nm UV irradiationat 16, 20, 24 and 48 hours for fluorescence by means of Digistore 2Image documentation system (CAMAG). Data are shown in Tables IVa, IVband IVc. Loops of bacterial cells, grown for 48 hours on Nutrient Agarplates containing 150 mg/l beta-D-galactosidase indicator and 100 mg/lIPTG, were directly streaked into wells of black clear bottom microtiterplates. Fluorescence scans were recorded on a Spectramax M5 (MolecularDevices). Data are shown in FIG. 2. Stained cells were shown to bedetectable under a standard fluorescence microscope.

IO staining is accompanied by significant fluorescence. While greenfluorescence observed appeared to be of transient nature, (caused byfluorescence of the active 1H-indol-3-yl signalogen) IO fluorescence atlonger wavelengths (yellow-red) was found to be persistent.

Conclusion: IO staining represents a potentially valuable method fordetection and isolation of microbial species providing a means of longwave fluorescence staining of live microbial colonies and a novel simpletool for fluorescence labeling of individual cells.

Example 16 Indication of Carbamate Hydrolysis

When 1-(2-benzoylphenyl)-1H-indol-3-yl ethyl carbamate (I32) wassubjected to short treatment with 1N aqueous NaOH solution or prolongedexposure to pig liver esterase a yellow precipitate was produced whichwas identified as 11-phenyl-10H-indolo[1,2-a]indol-10-one (IO19) by TLCcomparison with reference samples.

Apparently, 11-phenyl-10H-indolo[1,2-a]indol-10-imine (IO32), which isexpected to yield from the aldol condensation is rapidly hydrolyzed toIO19.

Conclusion: The concept of IO staining can be expanded to includeindicators producing 1-(2-benzoylphenyl)-1H-indol-3-amine (aS32) orsimilar active signalogens hence providing the potential design for longsought precipitating indicators for amino-peptidase enzymes.

Example 17 Indication of Disulfide Reduction

Bis[1-(2-acetylphenyl)-1H-indol-3-yl]-disulfane (I33, 2 mg) wasdissolved in methanol (0.5 ml). Tris(2-carboxyethyl)phosphinehydrochloride (TCEP, 20 mg) was added to the almost colorless solution.Upon subsequent addition of 1 N aqueous sodium hydroxide solution thesolution turned yellow.

Conclusion: (1) IO staining/indication is potentially useful fordetection reducing environments and (2) the concept of IO staining canpotentially be expanded to include indicators producing1-(2-acetylphenyl)-1H-indol-3-thiol (aS33) or similar activesignalogens.

TABLE Ia List of 1H-Indol-3-yl Indicators Chemical Structure ofIndicator (R₄ = H) Entry Code Indicator LG aS R₁ R₂ R₃ R₅ X R₆ R₇ R₈ R₉R₁₀ 1 I1a 1H-Indol-3-yl-beta-D- a 1 H H H H O — — — — —galactopyranoside 2 I1c 1H-Indol-3-yl acetate c 1 H H H H O — — — — — 3I2a 4-Chloro-1H-indol-3-yl-beta- a 2 Cl H H H O — — — — —D-galactopyranoside 4 I3a 6-Chloro-1H-indol-3-yl-beta- a 3 H H Cl H O —— — — — D-galactopyranoside 5 I3e 6-Chloro-1H-indol-3-yl-alpha- e 3 H HCl H O — — — — — D-glucopyranoside 6 I3f 6-Chloro-1H-indol-3-yl-beta- f3 H H Cl H O — — — — — D-glucuronide sodium salt 7 I3g6-Chloro-1H-indol-3-yl-N- g 3 H H Cl H O — — — — — acetyl-beta-D-galactosaminide 8 I4a 5-Bromo-4-chloro-1H-indol-3- a 4 Cl Br H H O — — —— — yl-beta-D-galactopyranoside 9 I4b 5-Bromo-3-(tert- b 4 Cl Br H H O —— — — — butyldimethylsilyloxy)-4- chloro-1H-indole 10 I4c5-Bromo-4-chloro-1H-indol-3- c 4 Cl Br H H O — — — — — yl acetate 11 I4d5-Bromo-4-chloro-1H-indol-3- d 4 Cl Br H H O — — — — — yl cholinephosphate 12 I4h 5-Bromo-4-chloro-1H-indol-3- h 4 Cl Br H H O — — — — —yl-beta-D-glucopyranoside 13 I5a 5-Bromo-6-chloro-1H-indol-3- a 5 H BrCl H O — — — — — yl-beta-D-galactopyranoside 14 I6- 1H-Indol-3-yl-ethylcarbamate 1) 6 H H H H N — — — — — 15 I8a 1-Phenyl-1H-indol-3-yl-beta- a8 H H H R₁₁ O H H H H H D-galactopyranoside 16 I9a6-Chloro-1-phenyl-1H-indol- a 9 H H Cl R₁₁ O H H H H H 3-yl-beta-D-galactopyranoside 17 I10a 1-[2-(Methoxy- a 10 H H H R₁₂ O H H H H OMecarbonyl)phenyl]-1H-indol-3- yl-beta-D-galactopyranoside 18 I11a6-Chloro-1-[2-(methoxy- a 11 H H Cl R₁₂ O H H H H OMecarbonyl)phenyl]-1H-indol-3- yl-beta-D-galactopyranoside 19 I12a5-Bromo-4-chloro-1-[2- a 12 Cl Br H R₁₂ O H H H H OMe(methoxycarbonyl)phenyl]- 1H-indol-3-yl-beta-D- galactopyranoside 20I13a 1-(2-Formylphenyl)-1H-indol- a 13 H H H R₁₂ O H H H H H3-yl-beta-D- galactopyranoside 21 I14a 1-(2-Acetylphenyl)-1H-indol- a 14H H H R₁₂ O H H H H Me 3-yl-beta-D- galactopyranoside 22 I15a1-(2-Acetylphenyl)-4-chloro- a 15 Cl H H R₁₂ O H H H H Me1H-indol-3-yl-beta-D- galactopyranoside 23 I16a1-(2-Acetylphenyl)-6-chloro- a 16 H H Cl R₁₂ O H H H H Me1H-indol-3-yl-beta-D- galactopyranoside 24 I17a1-(2-Acetylphenyl)-5-bromo- a 17 Cl Br H R₁₂ O H H H H Me4-chloro-1H-indol-3-yl-beta- D-galactopyranoside 25 I18a1-(2-Acetyl-5-methoxy- a 18 H H Cl R₁₂ O H OMe H H Mephenyl)-6-chloro-1H-indol-3- yl-beta-D-galactopyranoside 26 I19a1-(2-Benzoylphenyl)-1H- a 19 H H H R₁₂ O H H H H Phe indol-3-yl-beta-D-galactopyranoside 27 I19c 1-(2-Benzoylphenyl)-1H- c 19 H H H R₁₂ O H H HH Phe indol-3-yl acetate 28 I20a 1-(2-Benzoylphenyl)-4-chloro- a 20 Cl HH R₁₂ O H H H H Phe 1H-indol-3-yl-beta-D- galactopyranoside 29 I21a1-(2-Benzoylphenyl)-6-chloro- a 21 H H Cl R₁₂ O H H H H Phe1H-indol-3-yl-beta-D- galactopyranoside 30 I21e1-(2-Benzoylphenyl)-6-chloro- e 21 H H Cl R₁₂ O H H H H Phe1H-indol-3-yl-alpha-D- glucopyranoside 31 I21f1-(2-Benzoylphenyl)-6-chloro- f 21 H H Cl R₁₂ O H H H H Phe1H-indol-3-yl-beta-D- glucuronide sodium salt 32 I21g1-(2-Benzoylphenyl)-6-chloro- g 21 H H Cl R₁₂ O H H H H Phe1H-indol-3-yl-N-acetyl-beta- D-galactosaminide 33 I22a1-(2-Benzoylphenyl)-5- a 22 Cl Br H R₁₂ O H H H H Phebromo-4-chloro-1H-indol-3-yl- beta-D-galactopyranoside 34 I23a1-(2-Benzoyl-5-chlorphenyl)- a 23 H H Cl R₁₂ O H Cl H H Phe6-chloro-1H-indol-3-yl-beta- D-galactopyranoside 35 I24a1-(2-Benzoyl-5-methoxy- a 24 H H Cl R₁₂ O H OMe H H Phephenyl)-6-chloro-1H-indol-3- yl-beta-D-galactopyranoside 36 I25a1-[2-(2,4-Dimethoxy- a 25 H H H R₁₂ O H H H H DMPbenzoyl)phenyl]-1H-indol-3- yl-beta-D-galactopyranoside 37 I25c1-[2-(2,4-Dimethoxy-- c 25 H H H R₁₂ O H H H H DMPbenzoyl)phenyl]-1H-indol-3-yl acetate 38 I25d 1-[2-(2,4-Dimethoxy- d 25H H H R₁₂ O H H H H DMP benzoyl)phenyl]-1H-indol-3-yl choline phosphate39 I26a 1-[2-(2,4-Dimethoxy- a 26 Cl H H R₁₂ O H H H H DMPbenzoyl)phenyl]-4-chloro-1H- indol-3-yl-beta-D- galactopyranoside 40I27a 6-Chloro 1-[2-(2,4-dimethoxy- a 27 H H Cl R₁₂ O H H H H DMPbenzoyl)phenyl]-1H-indol-3- yl-beta-D-galactopyranoside 41 I28a5-Bromo-4-chloro-1-[2-(2,4- a 28 Cl Br H R₁₂ O H H H H DMPdimethoxybenzoyl)phenyl]- 1H-indol-3-yl-beta-D- galactopyranoside 42I29a 1-[2-(5-Carboxylfuranoyl)- a 29 H H Cl R₁₂ O H H H H CFurphenyl]-6-chloro-1H-indol-3- yl-beta-D-galactopyranoside 43 I30a6-Chloro-1-(2-furanoyl- a 30 H H Cl R₁₂ O H H H H Furphenyl)-1H-indol-3-yl-beta-D- galactopyranoside 44 I31a6-Chloro-1-[2-(N-methyl- a 31 H H Cl R₁₂ O H H H H NPyrpyrrole-2-carbonyl)phenyl]- 1H-indol-3-yl-beta-D- galactopyranoside 45I32- 1-(2-Benzoylphenyl)-1H- 1) 32 H H H R₁₂ N H H H H Phe indol-3-ylethyl carbamate 46 I33- Bis[1-(2-acetylphenyl)-1H- 2) 33 H H H R₁₂ S H HH H Me indol-3-yl]-disulfane 1) LG: —N—C═O-Ethyl (ethylcarbamate) 2) LG:—S—S— (disulfane) or isothiuronium iodide

TABLE Ib List of 1H-Indol-3-yl Active Signalogens Chemical Structure ofActive Signalogen Entry Code Active Signalogen R₁ R₂ R₃ R₄ R₅ X R₆ R₇ R₈R₉ R₁₀ 1 aS1 1H-Indol-3-ol H H H H H O — — — — — 2 aS24-Chloro-1H-indol-3-ol Cl H H H H O — — — — — 3 aS36-Chloro-1H-indol-3-ol H H Cl H H O — — — — — 4 aS45-Bromo-4-chloro-1H-indol-3- Cl Br H H H O — — — — — ol 5 aS55-Bromo-6-chloro-1H-indol-3- H Br Cl H H O — — — — — ol 6 aS61H-Indol-3-amine H H H H H N — — — — — 7 aS7 1H-Indol-3-thiol H H H H HS — — — — — 8 aS8 1-Phenyl-1H-indol-3-ol H H H H R₁₁ O H H H H H 9 aS96-Chloro-1-phenyl-1H-indol-3- H H Cl H R₁₁ O H H H H H ol 10 aS101-[2-(Methoxy- H H H H R₁₂ O H H H H OMe carbonyl)phenyl]-1H-indol-3-ol11 aS11 1-[2-(Methoxy- H H Cl H R₁₂ O H H H H OMecarbonyl)phenyl]-6-chloro-1H- indol-3-ol 12 aS12 5-Bromo-4-chloro-1-[2-Cl Br H H R₁₂ O H H H H OMe (methoxycarbonyl)phenyl)-1H- indol-3-ol 13aS13 1-(2-Formylphenyl)-1H-indol-3- H H H H R₁₂ O H H H H H ol 14 aS141-(2-Acetylphenyl)-1H-indol-3- H H H H R₁₂ O H H H H Me ol 15 aS151-(2-Acetylphenyl)-4-chloro- Cl H H H R₁₂ O H H H H Me 1H-indol-3-ol 16aS16 1-(2-Acetylphenyl)-6-chloro- H H Cl H R₁₂ O H H H H Me1H-indol-3-ol 17 aS17 1-(2-Acetylphenyl)-5-bromo-4- Cl Br H H R₁₂ O H HH H Me chloro-1H-indol-3-ol 18 aS18 1-(2-Acetyl-5-methoxyphenyl)- H H ClH R₁₂ O H OMe H H Me 6-chloro-1H-indol-3-ol 19 aS191-(2-Benzoylphenyl)-1H-indol- H H H H R₁₂ O H H H H Phe 3-ol 20 aS201-(2-Benzoylphenyl)-4-chloro- Cl H H H R₁₂ O H H H H Phe 1H-indol-3-ol21 aS21 1-(2-Benzoylphenyl)-6-chloro- H H Cl H R₁₂ O H H H H Phe1H-indol-3-ol 22 aS22 1-(2-Benzoylphenyl)-5-bromo- Cl Br H H R₁₂ O H H HH Phe 4-chloro-1H-indol-3-ol 23 aS23 1-(2-Benzoyl-5-chlorphenyl)-6- H HCl H R₁₂ O H Cl H H Phe chloro-1H-indol-3-ol 24 aS241-(2-Benzoyl-5-methoxy- H H Cl H R₁₂ O H OMe H H Phephenyl)-6-chloro-1H-indol-3-ol 25 aS25 1-[2-(2,4-Dimethoxy- H H H H R₁₂O H H H H DMP benzoyl)phenyl]-1H-indol-3-ol 26 aS264-Chloro-1-[2-(2,4-dimethoxy- Cl H H H R₁₂ O H H H H DMPbenzoyl)phenyl]-1H-indol-3-ol 27 aS27 6-Chloro-1-[2-(2,4-dimethoxy- H HCl H R₁₂ O H H H H DMP benzoyl)phenyl]-1H-indol-3-ol 28 aS285-Bromo-4-chloro-1-[2-(2,4- Cl Br H H R₁₂ O H H H H DMPdimethoxybenzoyl)phenyl]-1H- indol-3-ol 29 aS296-Chloro-1-[2-(5-carboxyl- H H Cl H R₁₂ O H H H H CFurfuranoyl)phenyl]-1H-indol-3-ol 30 aS30 6-Chloro-1-(2-furanoylphenyl)- HH Cl H R₁₂ O H H H H Fur 1H-indol-3-ol 31 aS31 6-Chloro-1-[2-(N-methyl-H H Cl H R₁₂ O H H H H NPyr pyrrole-2-carbonyl)phenyl]-1H- indol-3-ol 32aS32 1-(2-Benzoylphenyl)-1H-indol- H H H H R₁₂ N H H H H Phe 3-amine 33aS33 1-(2-Acetylphenyl)-1H-indol-3- H H H H R₁₂ S H H H H Me thiol

TABLE Ic List of Signalophores produced by 1H-indol-3-yl IndicatorSystems Chemical Structure of Signalophore (R₁₃ = R₁₀, OH) Entry CodeType Signalophore aS aR R₁ R₂ R₃ R₄ R₅ X R₆ R₇ R₈ R₉ R₁₃ 1 IN1 IN Indigo1 Air H H H H H O — — — — — 2 IN2 IN 4,4′-Dichloroindigo 2 Air Cl H H HH O — — — — — 3 IN3 IN 6,6′-Dichloroindigo 3 Air H H Cl H H O — — — — —4 IN4 IN 5,5′-Dibromo-4,4′- 4 Air Cl Br H H H O — — — — — dichloroindigo5 IN5 IN 5,5′-Dibromo-6,6′- 5 Air H Br Cl H H O — — — — — dichloroindigo6 IN8 IN 1,1′-Diphenylindigo 8 Air H H H H R₁₁ O H H H H H 7 IN9 IN6,6′-Dichloro-1,1- 9 Air H H Cl H R₁₁ O H H H H H diphenylindigo 8 IN10IN 1,1′-Di[2-(methoxy- 10 Air H H H H R₁₂ O H H H H OMecarbonyl)phenyl]indigo 9 IN11 IN 1,1′-Di[2-(methoxy- 11 Air H H Cl H R₁₂O H H H H OMe carbonyl)phenyl]-6,6′- dichloroindigo 10 IN12 IN1,1′-Di[2-(methoxy- 12 Air Cl Br H H R₁₂ O H H H H OMecarbonyl)phenyl]-5,5′- dibromo-4,4′- dichloroindigo 11 BI1 BI2-(4-Dimethylamino-2- 1 MDAB H H H H H O — — — — —methoxybenzylidene)indolin- 3-one 12 BI2 BI 4-Chloro-2-(4- 2 MDAB Cl H HH H O — — — — — dimethylamino-2- methoxybenzylidene)indolin- 3-one 13BI3 BI 6-Chloro-2-(4- 3 MDAB H H Cl H H O — — — — — dimethylamino-2-methoxybenzylidene)indolin- 3-one 14 BI4 BI 5-Bromo-4-chloro-2- 4 MDABCl Br H H H O — — — — — (4-dimethylamino-2- methoxybenzylidene)indolin-3-one 15 BI5 BI 5-Bromo-6-chloro-2- 5 MDAB H Br Cl H H O — — — — —(4-dimethylamino-2- methoxybenzylidene)indolin- 3-one 16 BI8 BI2-(4-Dimethylamino-2- 8 MDAB H H H H R₁₁ O H H H H Hmethoxybenzylidene)- 1-phenylindolin-3-one 17 BI9 BI 6-Chloro-2-(4- 9MDAB H H Cl H R₁₁ O H H H H H dimethylamino-2- methoxybenzylidene)-1-phenylindolin-3-one 18 BI10 BI 2-(4-Dimethylamino-2- 10 MDAB H H H HR₁₂ O H H H H OMe methoxybenzylidene)-1-(2-(methoxycarbonyl)phenyl)indolin- 3- one 19 BI11 BI 6-Chloro-2-(4-11 MDAB H H Cl H R₁₂ O H H H H OMe dimethylamino-2- methoxybenzylidene)-1-[2-(methoxy- carbonyl)- phenyl]indolin-3-one 20 BI12 BI5-Bromo-4-chloro-2- 12 MDAB Cl Br H H R₁₂ O H H H H OMe(4-dimethylamino-2- methoxybenzylidene)- 1-[2-(methoxy- carbonyl)-phenyl]indolin-3-one 21 BI6 BI 2-(4-Dimethylamino-2- 6 MDAB H H H H H N— — — — — methoxybenzylidene)indolin- 3-imine 22 BI7 BI2-(4-Dimethylamino-2- 7 MDAB H H H H H S — — — — —methoxybenzylidene)indolin- 3-thione 23 IO10 IO 11-Hydroxy-10H-indolo[1,10 — H H H H NA O H H H H OH 2-a]indol-10-one 24 IO11 IO7-Chloro-11-hydroxy- 11 — H H Cl H NA O H H H H OH10H-indolo[1,2-a]indol- 10-one 25 IO12 IO 8-Bromo-9-chloro-11- 12 — ClBr H H NA O H H H H OH hydroxy-10H-indolo- [1,2-a]indol-10-one 26 IO13IO 10H-Indolo[1,2-a]indol- 13 — H H H H NA O H H H H H 10-one 27 IO14 IO11-Methyl-10H-indolo- 14 — H H H H NA O H H H H Me [1,2-a]indol-10-one28 IO15 IO 9-Chloro-11-methyl- 15 — Cl H H H NA O H H H H Me10H-indolo[1,2-a]indol- 10-one 29 IO16 IO 7-Chloro-11-methyl- 16 — H HCl H NA O H H H H Me 10H-indolo[1,2-a]indol- 10-one 30 IO17 IO8-Bromo-9-chloro-11- 17 — Cl Br H H NA O H H H H Memethyl-10H-indolo[1,2- a]indol-10-one 31 IO18 IO 7-Chloro-11-methyl-3-18 — H H Cl H NA O H OMe H H Me methoxy-10H-indolo- [1,2-a]indol-10-one32 IO19 IO 11-Phenyl-10H-indolo- 19 — H H H H NA O H H H H Phe[1,2-a]indol-10-one 33 IO20 IO 9-Chloro-11-phenyl- 20 — Cl H H H NA O HH H H Phe 10H-indolo[1,2-a]indol- 10-one 34 IO21 IO 7-Chloro-11-phenyl-21 — H H Cl H NA O H H H H Phe 10H-indolo[1,2-a]indol- 10-one 35 IO22 IO8-Bromo-9-chloro-11- 22 — Cl Br H H NA O H H H H Phephenyl-10H-indolo[1,2- a]indol-10-one 36 IO23 IO 3,7-Dichloro-11- 23 — HH Cl H NA O H Cl H H Phe phenyl-10H-indolo[1,2- a]indol-10-one 37 IO24IO 7-Chloro-3-methoxy- 24 — H H Cl H NA O H OMe H H Phe11-phenyl-10H-indolo- [1,2-a]indol-10-one 38 IO25 IO 11-(2,4-Dimethoxy)-25 — H H H H NA O H H H H DMP phenyl-10H-indolo[1,2- a]indol-10-one 39IO27 IO 7-Chloro-11-(2,4- 27 — H H Cl H NA O H H H H DMPdimethoxy)phenyl- 10H-indolo[1,2-a]indol- 10-one 40 IO26 IO9-Chloro-11-(2,4- 26 — Cl H H H NA O H H H H DMP dimethoxy)phenyl-10H-indolo[1,2-a]indol- 10-one 41 IO28 IO 8-Bromo-9-chloro-11- 28 — ClBr H H NA O H H H H DMP (2,4-dimethoxy)phenyl- 10H-indolo[1,2-a]indol-10-one 42 IO29 IO 7-Chloro-11-(5- 29 — H H Cl H NA O H H H HCFur carboxyfuran-2-yl)- 10H-indolo[1,2-a]indol- 10-one 43 IO30 IO7-Chloro-11-(furan-2- 30 — H H Cl H NA O H H H H Fur yl)-10H-indolo[1,2-a]indol-10-one 44 IO32 IO 11-Phenyl-10H- 32 — H H H H NA N H H H H Pheindolo[1,2-a]indol-10- imine 45 IO33 IO 11-Metyl-10H-indolo- 33 — H H HH NA S H H H H Me [1,2-a]indol-10-thione 46 IO31 IO 7-Chloro-11-(N- 31 —H H Cl H NA O H H H H NPyr methylpyrrol-2-yl)- 10H-indolo[1,2-a]indol-10-one

TABLE IIa List of Aerobic 1H-Indol-3-yl Indicator Systems (no MDAB)Signalophore Entry Indicator/Active Signalogen Code aS aR LG ExternalStimulus BI IO IN 1 1H-Indol-3-yl-beta-D- I1a 1 Air a Enzyme: beta-D- NANA 1 galactopyranoside galactosidase 2 1H-Indol-3-yl acetate I1c 1 Air cEnzyme: Esterase; Ion: NA NA 1 Hydroxide 3 4-Chloro-1H-indol-3-yl-beta-I2a 2 Air a Enzyme: beta-D- NA NA 2 D-galactopyranoside galactosidase 46-Chloro-1H-indol-3-yl-beta- I3a 3 Air a Enzyme: beta-D- NA NA 3D-galactopyranoside galactosidase 5 6-Chloro-1H-indol-3-yl-alpha- I3e 3Air e Enzyme: alpha- NA NA 3 D-glucopyranoside glucosidase 66-Chloro-1H-indol-3-yl-beta- I3f 3 Air f Enzyme: beta-D- NA NA 3D-glucuronide sodium salt glucuronidase 7 6-Chloro-1H-indol-3-yl-N- I3g3 Air g Enzyme: Galactosamidase NA NA 3 acetyl-beta-D- galactosaminide 85-Bromo-4-chloro-1H-indol-3- I4a 4 Air a Enzyme: beta-D- NA NA 4yl-beta-D-galactopyranoside galactosidase 9 5-Bromo-3-(tert- I4b 4 Air bIon: Fluoride NA NA 4 butyldimethylsilyloxy)-4- chloro-1H-indole 105-Bromo-4-chloro-1H-indol-3- I4c 4 Air c Enzyme: Esterase; Ion: NA NA 4yl acetate Hydroxide 11 5-Bromo-4-chloro-1H-indol-3- I4d 4 Air d Enzyme:Phospholipase c NA NA 4 yl choline phosphate 125-Bromo-4-chloro-1H-indol-3- I4h 4 Air h Enzyme: beta-D- NA NA 4yl-beta-D-glucopyranoside glucosidase 13 5-Bromo-6-chloro-1H-indol-3-I5a 5 Air a Enzyme: beta-D- NA NA 4 yl-beta-D-galactopyranosidegalactosidase 14 1H-Indol-3-yl-ethyl carbamate I6 6 Air 1) Enzyme:Esterase; Ion: NA NA * Hydroxide 15 (1H-Indol-3-thiol) NA 7 Air 2) — NANA — 16 1-Phenyl-1H-indol-3-yl-beta- I8a 8 Air a Enzyme: beta-D- NA NA 8D-galactopyranoside galactosidase 17 6-Chloro-1-phenyl-1H-indol- I9a 9Air a Enzyme: beta-D- NA NA 9 3-yl-beta-D- galactosidasegalactopyranoside 18 1-[2-(Methoxy- I10a 10 Air a Enzyme: beta-D- NA 10(10)  carbonyl)phenyl]-1H-indol-3- galactosidaseyl-beta-D-galactopyranoside 19 6-Chloro-1-[2-(methoxy- I11a 11 Air aEnzyme: beta-D- NA 11 (11)  carbonyl)phenyl]-1H-indol-3- galactosidaseyl-beta-D-galactopyranoside 20 5-Bromo-4-chloro-1-[2- I12a 12 Air aEnzyme: beta-D- NA 12 (12)  (methoxycarbonyl)phenyl]- galactosidase1H-indol-3-yl-beta-D- galactopyranoside 21 1-(2-Formylphenyl)-1H-indol-I13a 13 Air a Enzyme: beta-D- NA 13 — 3-yl-beta-D- galactosidasegalactopyranoside 22 1-(2-Acetylphenyl)-1H-indol- I14a 14 Air a Enzyme:beta-D- NA 14 — 3-yl-beta-D- galactosidase galactopyranoside 231-(2-Acetylphenyl)-4-chloro- I15a 15 Air a Enzyme: beta-D- NA 15 —1H-indol-3-yl-beta-D- galactosidase galactopyranoside 241-(2-Acetylphenyl)-6-chloro- I16a 16 Air a Enzyme: beta-D- NA 16 —1H-indol-3-yl-beta-D- galactosidase galactopyranoside 251-(2-Acetylphenyl)-5-bromo- I17a 17 Air a Enzyme: beta-D- NA 17 —4-chloro-1H-indol-3-yl-beta- galactosidase D-galactopyranoside 261-(2-Acetyl-5- I18a 18 Air a Enzyme: beta-D- NA 18 —methoxyphenyl)-6-chloro-1H- galactosidase indol-3-yl-beta-D-galactopyranoside 27 1-(2-Benzoylphenyl)-1H- I19a 19 Air a Enzyme:beta-D- NA 19 — indol-3-yl-beta-D- galactosidase galactopyranoside 281-(2-Benzoylphenyl)-1H- I19c 19 Air c Enzyme: Esterase; Ion: NA 19 —indol-3-yl acetate Hydroxide 29 1-(2-Benzoylphenyl)-4- I20a 20 Air aEnzyme: beta-D- NA 20 — chloro-1H-indol-3-yl-beta-D- galactosidasegalactopyranoside 30 1-(2-Benzoylphenyl)-6- I21a 21 Air a Enzyme:beta-D- NA 21 — chloro-1H-indol-3-yl-beta-D- galactosidasegalactopyranoside 31 1-(2-Benzoylphenyl)-6- I21e 21 Air e Enzyme: alpha-NA 21 — chloro-1H-indol-3-yl-alpha-D- glucosidase glucopyranoside 321-(2-Benzoylphenyl)-6- I21f 21 Air f Enzyme: beta-D- NA 21 —chloro-1H-indol-3-yl-beta-D- glucuronidase glucuronide sodium salt 331-(2-Benzoylphenyl)-6- I21g 21 Air g Enzyme: Galactosamidase NA 21 —chloro-1H-indol-3-yl-N-acetyl- beta-D-galactosaminide 341-(2-Benzoylphenyl)-5- I22a 22 Air a Enzyme: beta-D- NA 22 —bromo-4-chloro-1H-indol-3-yl- galactosidase beta-D-galactopyranoside 351-(2-Benzoyl-5-chlorphenyl)- I23a 23 Air a Enzyme: beta-D- NA 23 —6-chloro-1H-indol-3-yl-beta- galactosidase D-galactopyranoside 361-(2-Benzoyl-5-methoxy- I24a 24 Air a Enzyme: beta-D- NA 24 —phenyl)-6-chloro-1H-indol-3- galactosidase yl-beta-D-galactopyranoside37 1-[2-(2,4-Dimethoxy- I25a 25 Air a Enzyme: beta-D- NA 25 —benzoyl)lphenyl]-1H-indol-3- galactosidase yl-beta-D-galactopyranoside38 1-[2-(2,4-Dimethoxy- I25c 25 Air c Enzyme: Esterase; Ion: NA 25 —benzoyl)phenyl]-1H-indol-3-yl Hydroxide acetate 39 1-[2-(2,4-Dimethoxy-I25d 25 Air d Enzyme: Phospholipase c NA 25 —benzoyl)phenyl]-1H-indol-3-yl choline phosphate 406-Chloro-1-[2-(2,4-dimethoxy- I27a 26 Air a Enzyme: beta-D- NA 26 —benzoyl)phenyl]-1H-indol-3- galactosidase yl-beta-D-galactopyranoside 414-Chloro-1-[2-(2,4-dimethoxy- I26a 27 Air a Enzyme: beta-D- NA 27 —benzoyl)phenyl]-1H-indol-3- galactosidase yl-beta-D-galactopyranoside 425-Bromo-4-chloro-1-[2-(2,4- I28a 28 Air a Enzyme: beta-D- NA 28 —dimethoxybenzoyl)phenyl]- galactosidase 1H-indol-3-yl-beta-D-galactopyranoside 43 1-[2-(5-Carboxyl- I29a 29 Air a Enzyme: beta-D- NA29 — furanoyl)phenyl]-6-chloro-1H- galactosidase indol-3-yl-beta-D-galactopyranoside 44 6-Chloro-1-(2-furanoyl- I30a 30 Air a Enzyme:beta-D- NA 30 — phenyl)-1H-indol-3-yl-beta-D- galactosidasegalactopyranoside 45 6-Chloro-1-[2-(N-methyl- I31a 31 Air a Enzyme:beta-D- NA 31 — pyrrole-2-carbonyl)phenyl]- galactosidase1H-indol-3-yl-beta-D- galactopyranoside 46 1-(2-Benzoylphenyl)-1H- I3232 Air 1) Enzyme: Esterase; Ion: NA 32 — indol-3-yl ethyl carbamateHydroxide 47 Bis[1-(2-acetylphenyl)-1H- I33 33 Air 2) Reducing Agent:TCEP NA 33 — indol-3-yl]-disulfane NP: Not Performed NA: Not Applicable*: dark precipitate 1) LG: —N—C═O-Ethyl (ethylcarbamate) 2) LG:—S—S-(disulfane) or isothiuronium iodide

TABLE IIb List of Aerobic 1H-Indol-3-yl/MDAB Indicator SystemsSignalophore Entry Indicator/Active Signalogen Code aS aR LG ExternalStimulus BI IO IN 1 1H-Indol-3-yl-beta-D- I1a 1 Air/MDAB a Enzyme:beta-D- (1) NA 1 galactopyranoside galactosidase 2 1H-Indol-3-yl acetateI1c 1 Air/MDAB c Enzyme: Esterase; NP NA NP Ion: Hydroxide 34-Chloro-1H-indol-3-yl-beta- I2a 2 Air/MDAB a Enzyme: beta-D- (2) NA 2D-galactopyranoside galactosidase 4 6-Chloro-1H-indol-3-yl-beta- I3a 3Air/MDAB a Enzyme: beta-D- (3) NA 3 D-galactopyranoside galactosidase 56-Chloro-1H-indol-3-yl-alpha- I3e 3 Air/MDAB e Enzyme: alpha- NP NA NPD-glucopyranoside glucosidase 6 6-Chloro-1H-indol-3-yl-beta- I3f 3Air/MDAB f Enzyme: beta-D- NP NA NP D-glucuronide sodium saltglucuronidase 7 6-Chloro-1H-indol-3-yl-N- I3g 3 Air/MDAB g Enzyme:Galactosamidase NP NA NP acetyl-beta-D- galactosaminide 85-Bromo-4-chloro-1H-indol-3- I4a 4 Air/MDAB a Enzyme: beta-D- (4) NA 4yl-beta-D-galactopyranoside galactosidase 9 5-Bromo-3-(tert- I4b 4Air/MDAB b Ion: Fluoride (4) NA 4 butyldimethylsilyloxy)-4-chloro-1H-indole 10 5-Bromo-4-chloro-1H-indol-3- I4c 4 Air/MDAB cEnzyme: Esterase; (4) NA 4 yl acetate Ion: Hydroxide 115-Bromo-4-chloro-1H-indol-3- I4d 4 Air/MDAB d Enzyme: Phospholipase c NPNP NP yl choline phosphate 12 5-Bromo-4-chloro-1H-indol-3- I4h 4Air/MDAB h Enzyme: beta-D- NP NP NP yl-beta-D-glucopyranosideglucosidase 13 5-Bromo-6-chloro-1H-indol-3- I5a 5 Air/MDAB a Enzyme:beta-D- (4) NA 4 yl-beta-D-galactopyranoside galactosidase 141H-Indol-3-yl-ethyl carbamate I6 6 Air/MDAB 1) Enzyme: Esterase; * NA —Ion: Hydroxide 15 (1H-Indol-3-thiol) NA 7 Air/MDAB 2) Ion: Hydroxyde (6)NA — 16 1-Phenyl-1H-indol-3-yl-beta- I8a 8 Air/MDAB a Enzyme: beta-D- NPNP NP D-galactopyranoside galactosidase 17 6-Chloro-1-phenyl-1H-indol-3-I9a 9 Air/MDAB a Enzyme: beta-D- 9 NA (9) yl-beta-D-galactopyranosidegalactosidase 18 1-[2-(Methoxy- I10a 10 Air/MDAB a Enzyme: beta-D- — 10— carbonyl)phenyl]-1H-indol-3- galactosidase yl-beta-D-galactopyranoside19 6-Chloro-1-[2-(methoxy- I11a 11 Air/MDAB a Enzyme: beta-D- 11  (10) —carbonyl)phenyl]-1H-indol-3- galactosidase yl-beta-D-galactopyranoside20 5-Bromo-4-chloro-1-[2- I12a 12 Air/MDAB a Enzyme: beta-D- — 12 (12) (methoxycarbonyl)phenyl]-1H- galactosidase indol-3-yl-beta-D-galactopyranoside 21 1-(2-Formylphenyl)-1H-indol- I13a 13 Air/MDAB aEnzyme: beta-D- — 13 (13)  3-yl-beta-D-galactopyranoside galactosidase22 1-(2-Acetylphenyl)-1H-indol-3- I14a 14 Air/MDAB a Enzyme: beta-D- —14 — yl-beta-D-galactopyranoside galactosidase 231-(2-Acetylphenyl)-4-chloro- I15a 15 Air/MDAB a Enzyme: beta-D- — 15 —1H-indol-3-yl-beta-D- galactosidase galactopyranoside 241-(2-Acetylphenyl)-6-chloro- I16a 16 Air/MDAB a Enzyme: beta-D- — 16 —1H-indol-3-yl-beta-D- galactosidase galactopyranoside 251-(2-Acetylphenyl)-5-bromo-4- I17a 17 Air/MDAB a Enzyme: beta-D- — 17 —chloro-1H-indol-3-yl-beta-D- galactosidase galactopyranoside 261-(2-Acetyl-5-methoxy- I18a 18 Air/MDAB a Enzyme: beta-D- — 18 —phenyl)-6-chloro-1H-indol-3- galactosidase yl-beta-D-galactopyranoside27 1-(2-Benzoylphenyl)-1H-indol- I19a 19 Air/MDAB a Enzyme: beta-D- — 19— 3-yl-beta-D-galactopyranoside galactosidase 281-(2-Benzoylphenyl)-1H-indol- I19c 19 Air/MDAB c Enzyme: Esterase; NP NPNP 3-yl acetate Ion: Hydroxide 29 1-(2-Benzoylphenyl)-4-chloro- I20a 20Air/MDAB a Enzyme: beta-D- — 20 — 1H-indol-3-yl-beta-D- galactosidasegalactopyranoside 30 1-(2-Benzoylphenyl)-6-chloro- I21a 21 Air/MDAB aEnzyme: beta-D- — 21 — 1H-indol-3-yl-beta-D- galactosidasegalactopyranoside 31 1-(2-Benzoylphenyl)-6-chloro- I21e 21 Air/MDAB eEnzyme: alpha- NP NP NP 1H-indol-3-yl-alpha-D- glucosidaseglucopyranoside 32 1-(2-Benzoylphenyl)-6-chloro- I21f 21 Air/MDAB fEnzyme: beta-D- NP NP NP 1H-indol-3-yl-beta-D- glucuronidase glucuronidesodium salt 33 1-(2-Benzoylphenyl)-6-chloro- I21g 21 Air/MDAB g Enzyme:Galactosamidase NP NP NP 1H-indol-3-yl-N-acetyl-beta-D- galactosaminide34 1-(2-Benzoylphenyl)-5-bromo- I22a 22 Air/MDAB a Enzyme: beta-D- — 22— 4-chloro-1H-indol-3-yl-beta-D- galactosidase galactopyranoside 351-(2-Benzoyl-5-chlorophenyl)- I23a 23 Air/MDAB a Enzyme: beta-D- — 23 —6-chloro-1H-indol-3-yl-beta-D- galactosidase galactopyranoside 361-(2-Benzoyl-5-methoxy- I24a 24 Air/MDAB a Enzyme: beta-D- — 24 —phenyl)-6-chloro-1H-indol-3- galactosidase yl-beta-D-galactopyranoside37 1-[2-(2,4-Dimethoxy- I25a 25 Air/MDAB a Enzyme: beta-D- — 25 —benzoyl)phenyl]-1H-indol-3-yl- galactosidase beta-D-galactopyranoside 381-[2-(2,4-Dimethoxy- I25c 25 Air/MDAB c Enzyme: Esterase; NP NP NPbenzoyl)phenyl]-1H-indol-3-yl Ion: Hydroxide acetate 391-[2-(2,4-Dimethoxy- I25d 25 Air/MDAB d Enzyme: Phospholipase c NP NP NPbenzoyl)phenyl]-1H-indol-3-yl choline phosphate 40 1-[2-(2,4-Dimethoxy-I27a 27 Air/MDAB a Enzyme: beta-D- — 27 — benzoyl)phenyl]-6-chloro-1H-galactosidase indol-3-yl-beta-D- galactopyranoside 411-[2-(2,4-Dimethoxy- I26a 26 Air/MDAB a Enzyme: beta-D- — 26 —benzoyl)phenyl]-4-chloro-1H- galactosidase indol-3-yl-beta-D-galactopyranoside 42 5-Bromo-4-chloro-1-[2-(2,4- I28a 28 Air/MDAB aEnzyme: beta-D- — 28 — dimethoxybenzoyl)phenyl]- galactosidase1H-indol-3-yl-beta-D- galactopyranoside 43 6-Chloro-1-[2-(5-carboxyl-I29a 29 Air/MDAB a Enzyme: beta-D- — 29 — furanoyl)phenyl)-1H-indol-3-galactosidase yl-beta-D-galactopyranoside 44 6-Chloro-1-(2-furanoyl-I30a 30 Air/MDAB a Enzyme: beta-D- — 30 — phenyl)-1H-indol-3-yl-beta-D-galactosidase galactopyranoside 45 6-Chloro-1-[2-(N-methyl- I31a 31Air/MDAB a Enzyme: beta-D- NP NP NP pyrrole-2-carbonyl)phenyl]-galactosidase 1H-indol-3-yl-beta-D- galactopyranoside 461-(2-Benzoylphenyl)-1H-indol- I32 32 Air/MDAB 1) Enzyme: Esterase; NP NPNP 3-yl ethyl carbamate Ion: Hydroxide 47 Bis(1-(2-acetylphenyl)-1H- I3333 Air/MDAB 2) Reducing Agent: NP NP NP indol-3-yl]-disulfane TCEP NP:Not Performed NA: Not Applicable *: yellow-brown solution 1) LG:—N—C═O-Ethyl (ethylcarbamate) 2) LG: —S—S-(disulfane) or isothiuroniumiodide

TABLE IIc List of Anaerobic 1H-Indol-3-yl/MDAB Indicator SystemsSignalophore Entry Indicator/(active Signalogen) Code aS aR LG ExternalStimulus BI IO IN 1 1H-Indol-3-yl-beta-D- I1a 1 MDAB a Enzyme: beta-D- 1NA — galactopyranoside galactosidase 2 1H-Indol-3-yl acetate I1c 1 MDABc Enzyme: Esterase; 1 NA — Ion: Hydroxide 34-Chloro-1H-indol-3-yl-beta-D- I2a 2 MDAB a Enzyme: beta-D- 2 NA —galactopyranoside galactosidase 4 6-Chloro-1H-indol-3-yl-beta-D- I3a 3MDAB a Enzyme: beta-D- 3 NA — galactopyranoside galactosidase 56-Chloro-1H-indol-3-yl-alpha-D- I3e 3 MDAB e Enzyme: alpha- NP NP NPglucopyranoside glucosidase 6 6-Chloro-1H-indol-3-yl-beta-D- I3f 3 MDABf Enzyme: beta-D- NP NP NP glucuronide sodium salt glucuronidase 76-Chloro-1H-indol-3-yl-N-acetyl- I3g 3 Air/MDAB g Enzyme: Galacto- NP NPNP beta-D-galactosaminide samidase 8 5-Bromo-4-chloro-1H-indol-3-yl- I4a4 MDAB a Enzyme: beta-D- 4 NA — beta-D-galactopyranoside galactosidase 95-Bromo-3-(tert-butyldimethyl- I4b 4 MDAB b Ion: Fluoride 4 NA —silyloxy)-4-chloro-1H-indole 10 5-Bromo-4-chloro-1H-indol-3-yl I4c 4MDAB c Enzyme: Esterase 4 NA — acetate 11 5-Bromo-4-chloro-1H-indol-3-ylI4d 4 MDAB d Enzyme: Phospholipase c NP NP NP choline phosphate 125-Bromo-4-chloro-1H-indol-3-yl- I4h 4 Air/MDAB h Enzyme: beta-D- NP NPNP beta-D-glucopyranoside glucosidase 13 5-Bromo-6-chloro-1H-indol-3-yl-I5a 5 MDAB a Enzyme: beta-D- 4 NA — beta-D-galactopyranosidegalactosidase 14 1H-Indol-3-yl-ethyl carbamate I6 6 MDAB — — NA * 15(1H-Indol-3-thiol) NA 7 MDAB — 6 NA — 16 1-Phenyl-1H-indol-3-yl-beta-D-I8a 8 MDAB a Enzyme: beta-D- NP NP NP galactopyranoside galactosidase 176-Chloro-1-phenyl-1H-indol-3-yl- I9a 9 MDAB a Enzyme: beta-D- 9 NA —beta-D-galactopyranoside galactosidase 18 1-[2-(Methoxycarbonyl)phenyl]-I10a 10 MDAB a Enzyme: beta-D- — 10 — 1H-indol-3-yl-beta-D-galactosidase galactopyranoside 19 6-Chloro-1-[2-(methoxy- I11a 11 MDABa Enzyme: beta-D- 11  11 — carbonyl)phenyl]-1H-indol-3-yl- galactosidasebeta-D-galactopyranoside 20 5-Bromo-4-chloro-1-[2-(methoxy- I12a 12 MDABa Enzyme: beta-D- — 12 — carbonyl)phenyl]-1H-indol-3-yl- galactosidasebeta-D-galactopyranoside 21 1-(2-Formylphenyl)-1H-indol-3-yl- I13a 13MDAB a Enzyme: beta-D- — 13 — beta-D-galactopyranoside galactosidase 221-(2-Acetylphenyl)-1H-indol-3-yl- I14a 14 MDAB a Enzyme: beta-D- — 14 —beta-D-galactopyranoside galactosidase 231-(2-Acetylphenyl)-4-chloro-1H- I15a 15 MDAB a Enzyme: beta-D- — 15 —indol-3-yl-beta-D- galactosidase galactopyranoside 241-(2-Acetylphenyl)-6-chloro-1H- I16a 16 MDAB a Enzyme: beta-D- — 16 —indol-3-yl-beta-D- galactosidase galactopyranoside 251-(2-Acetylphenyl)-5-bromo-4- I17a 17 MDAB a Enzyme: beta-D- — 17 —chloro-1H-indol-3-yl-beta-D- galactosidase galactopyranoside 261-(2-Acetyl-5-methoxyphenyl)-6- I18a 18 MDAB a Enzyme: beta-D- — 18 —chloro-1H-indol-3-yl-beta-D- galactosidase galactopyranoside 271-(2-Benzoylphenyl)-1H-indol-3- I19a 19 MDAB a Enzyme: beta-D- — 19 —yl-beta-D-galactopyranoside galactosidase 281-(2-Benzoylphenyl)-1H-indol-3-yl I19c 19 MDAB c Enzyme: Esterase NP NPNP acetate 29 1-(2-Benzoylphenyl)-4-chloro-1H- I20a 20 MDAB a Enzyme:beta-D- — 20 — indol-3-yl-beta-D- galactosidase galactopyranoside 301-(2-Benzoylphenyl)-6-chloro-1H- I21a 21 MDAB a Enzyme: beta-D- — 21 —indol-3-yl-beta-D- galactosidase galactopyranoside 311-(2-Benzoylphenyl)-6-chloro-1H- I21e 21 MDAB e Enzyme: alpha- NP NP NPindol-3-yl-alpha-D- glucosidase glucopyranoside 321-(2-Benzoylphenyl)-6-chloro-1H- I21f 21 MDAB f Enzyme: beta-D- NP NP NPindol-3-yl-beta-D-glucuronide glucuronidase sodium salt 331-(2-Benzoylphenyl)-6-chloro-1H- I21g 21 MDAB g Enzyme: Galacto- NP NPNP indol-3-yl-N-acetyl-beta-D- samidase galactosaminide 341-(2-Benzoylphenyl)-5-bromo-4- I22a 22 MDAB a Enzyme: beta-D- — 22 —chloro-1H-indol-3-yl-beta-D- galactosidase galactopyranoside 351-(2-Benzoyl-5-chlorophenyl)-6- I23a 23 MDAB a Enzyme: beta-D- — 23 —chloro-1H-indol-3-yl-beta-D- galactosidase galactopyranoside 361-(2-Benzoyl-5-methoxyphenyl)- I24a 24 MDAB a Enzyme: beta-D- — 24 —6-chloro-1H-indol-3-yl-beta-D- galactosidase galactopyranoside 371-[2-(2,4-Dimethoxy- I25a 25 MDAB a Enzyme: beta-D — 25 —benzoyl)lphenyl]-1H-indol-3-yl- galactosidase beta-D-galactopyranoside38 1-[2-(2,4-Dimethoxy- I25c 25 MDAB c Enzyme: Esterase NP NP NPbenzoyl)phenyl]-1H-indol-3-yl acetate 39 1-[2-(2,4-Dimethoxy- I25d 25MDAB d Enzyme: Phospholipase c NP NP NP benzoyl)phenyl]-1H-indol-3-ylcholine phosphate 40 1-[2-(2,4-Dimethoxy- I27a 27 MDAB a Enzyme: beta-D-— 27 — benzoyl)phenyl]-6-chloro-1H- galactosidase indol-3-yl-beta-D-galactopyranoside 41 1-[2-(2,4-Dimethoxy- I26a 26 MDAB a Enzyme: beta-D-— 26 — benzoyl)phenyl]-4-chloro-1H- galactosidase indol-3-yl-beta-D-galactopyranoside 42 1-[2-(2,4-Dimethoxy- I28a 28 MDAB a Enzyme: beta-D-— 28 — benzoyl)phenyl]-5-bromo-4- galactosidasechloro-1H-indol-3-yl-beta-D- galactopyranoside 431-[2-(5-Carboxylfuranoyl)phenyl]- I29a 29 MDAB a Enzyme: beta-D- — 29 —6-chloro-1H-indol-3-yl-beta-D- galactosidase galactopyranoside 446-Chloro-1-(2-furanoylphenyl)-1H- I30a 30 MDAB a Enzyme: beta-D- — 30 —indol-3-yl-beta-D- galactosidase galactopyranoside 456-Chloro-1-[2-(N-methylpyrrole-2- I31a 31 MDAB a Enzyme: beta-D- NP NPNP carbonyl)phenyl]-1H-indol-3-yl- galactosidasebeta-D-galactopyranoside 46 1-(2-Benzoylphenyl)-1H-indol-3-yl I32 32MDAB Enzyme: Esterase NP NP NP ethyl carbamate 47Bis(1-(2-acetylphenyl)-1H-indol-3- I33 33 MDAB Reducing Agent: NP NP NPyl]-disulfane TCEP NP: Not Performed NA: Not Applicable *: yellow-brownsolution 1) LG: —N—C═O-Ethyl (ethylcarbamate) 2) LG: —S—S-(disulfane) orisothiuronium iodide

TABLE III 2-Benzylideneindoline (MDAB) staining of colonies ofbeta-D-galactosidase positive/negative bacteria microaerobic (anaerobic)Entry Indicator aR Equivalents Species eS aerobic conditions conditionsb-gal TSAYE with 40 mg/100 ml TSAYE with 40 mg/100 ml of indicator ofindicator 1 I4a — NA Citrobacter + Blue green (dark colored Green (lightcolored colonies) freundii colonies) with no ring with no ring 2 I4a DAB2:1 Citrobacter + Blue green (medium colored Cream colored coloniesfreundii colonies) with no ring with no ring 3 I4a DAB 1:1 Citrobacter +Blue green (light colored Cream colored colonies freundii colonies) withclear ring with no ring 4 I4a MDAB 2:1 Citrobacter + Blue green (darkcolored Green (light colored colonies) freundii colonies) with lightring with no ring 5 I4a MDAB 1:1 Citrobacter + Blue green (light coloredPink (light colored colonies) freundii colonies) with pale red hue withno ring 6 I4a — NA Escherichia coli + Blue green (very dark Green (lightcolored colonies) colored colonies) with no with no ring ring 7 I4a DAB2:1 Escherichia coli + Blue green (very dark Pale green (very lightcolored colonies) with no colored colonies) with no ring ring 8 I4a DAB1:1 Escherichia coli + Blue green (very dark Cream colored coloniescolored colonies) with no with no ring ring 9 I4a MDAB 2:1 Escherichiacoli + Blue green (very dark Green (light colored colonies) coloredcolonies) with no with cream ring ring 10 I4a MDAB 1:1 Escherichiacoli + Blue green (very dark Pink (medium colored colored colonies) withwhite colonies) with cream ring ring 11 I4a MDAB 1:1 Salmonella − Creamcolored colonies Cream colored colonies manhattan 12 I4a MDAB 1:1Salmonella − Cream colored colonies Cream colored colonies manhattan 13I5a — NA Citrobacter + Violet (dark colored colonies) Pale violet (lightcolored freundii with a clear ring colonies) with no ring 14 I5a DAB 2:1Citrobacter + Violet (medium colored Cream colored colonies freundiicolonies) with a clear ring with no ring 15 I5a DAB 1:1 Citrobacter +Violet (light medium colored Cream colored colonies freundii colonies)with a white ring with no ring 16 I5a MDAB 2:1 Citrobacter + Violet(dark colored colonies) Violet (light colored colonies) freundii with aclear ring with a clear ring 17 I5a MDAB 1:1 Citrobacter + Violet(medium colored Violet (medium colored freundii colonies) with a clearring colonies) with a clear ring 18 I5a — NA Salmonella − Cream coloredcolonies Cream colored colonies manhattan 19 I5a — NA Escherichia coli +Violet (very dark colored Pale violet (light colored colonies) with noring colonies) with no ring 20 I5a DAB 2:1 Escherichia coli + Violet(very dark colored Cream colored colonies colonies) with no ring with noring 21 I5a DAB 1:1 Escherichia coli + Violet (medium colored Creamcolored colonies colonies) with a clear ring with no ring 22 I5a MDAB2:1 Escherichia coli + Violet (very dark colored Violet (light coloredcolonies) colonies) with no ring with a clear ring 23 I5a MDAB 1:1Escherichia coli + Violet (very dark colored Violet (medium coloredcolonies) with no ring colonies) with a clear ring 24 I5a — NASalmonella − Cream colored colonies Cream colored colonies manhattan 25I5a MDAB 1:1 Salmonella − Cream colored colonies Cream colored coloniesmanhattan 26 I9a — NA Escherichia coli + Cream to olive colored —colonies 27 I9a MDAB 1:1 Escherichia coli + Red colored colonies — 28I9a MDAB 1:1 Salmonella − Cream colored colonies — enteritidis 29 I10a —NA Escherichia coli + Cream to olive colored — colonies 30 I10a MDAB 1:1Escherichia coli + Cream to olive colored colonies 31 I10a MDAB 1:1Salmonella − Cream colored colonies — enteritidis 32 I11a — NAEscherichia coli + Cream to olive colored — colonies 33 I11a MDAB 1:1Escherichia coli + Red to brown colored colonies 34 I11a MDAB 1:1Salmonella − Cream colored colonies — enteritidis 35 I12a — NAEscherichia coli + Cream to olive colored — colonies 36 I12a MDAB 1:1Escherichia coli + Brown colored colonies 37 I12a MDAB 1:1 Salmonella −Cream colored colonies — enteritidis DAB: 4-(Dimethylamino)benzaldehydeMDAB: 2-Methoxy-4-(dimethylamino)benzaldehyde b-gal:beta-D-galactosidase −: absent +: present

TABLE IVa Color, fluorescence and localization of various IO stains onmicrobial plating media Bacterial colonies after 24 h on Nutrient Agarcontaining 150 mg/l substrate and 100 mg/l IPTG E. coli S. enteritidiseS: b-gal + − I19a 1-(2-Benzoylphenyl)-1H-indol-3-yl-β-D- Cyellow-orange + − galactopyranoside F yellow + − L + NA G + + I17a1-(2-Acetylphenyl)-5-bromo-4-chloro- C yellow-orange + −1H-indol-3-yl-β-D-galactopyranoside F — − − L + NA G + + I20a1-(2-Benzoylphenyl)-4-chloro-1H-indol- C orange +/− −3-yl-β-D-galactopyranoside F orange +/− − L cryst. NA G + + I13a1-(2-Formylphenyl)-1H-indol-3-yl-β-D- C cream-orange +/− −galactopyranoside F — − − L + G + + I10a1-[2-(Methoxycarbonyl)phenyl]-1H- C yellow-green + −indol-3-yl-β-D-galactopyranoside F green + − L − NA G + + I28a5-Bromo-4-chloro-1-[2-(2,4-dimethoxy- C orange + −benzoyl)phenyl]-1H-indol-3-yl-β-D- F (red) +/− − galactopyranoside L +NA G + + I26a 4-Chloro-1-[2-(2,4-dimethoxy- C yellow-orange + −benzoyl)phenyl]-1H-indol-3-yl-β-D- F orange +/− − galactopyranoside L +NA G + + I12a 5-Bromo-4-chloro-1-[2-(methoxy- C yellow-green +/− −carbonyl)phenyl]-1H-indol-3-yl-β-D- F green +/− − galactopyranoside L −NA G + + I11a 6-Chloro-1-[2-(methoxy- C yellow-green + −carbonyl)phenyl]-1H-indol-3-yl-β-D- F green + − galactopyranoside L − NAG + + I29a 6-Chloro-1-[2-(5-carboxyl- C red + −furanoyl)phenyl]-1H-indol-3-yl-β-D- F — − − galactopyranoside L − G + +I30a 6-Chloro-1-(2-furanoylphenyl)-1H- C dark orange ++ −indol-3-yl-β-D-galactopyranoside F orange-red +/− − L +/− G + + I23a6-Chloro-1-(2-benzoyl-5-chlorophenyl)- C yellow + NA1H-indol-3-yl-β-D-galactopyranoside F green-yellow + NA L + NA G + NAI18a 1-(2-Acetyl-5-methoxyphenyl)-1H- C light yellow + −indol-3-yl-β-D-galactopyranoside F green +/− − L +/− NA G + + I24a1-(2-Benzoyl-5-methoxyphenyl)-1H- C dark yellow + −indol-3-yl-β-D-galactopyranoside F yellow-green + − L +/− NA G + + I14a1-(2-Acetylphenyl)-1H-indol-3-yl-β-D- C light yellow + −galactopyranoside F — − − L + NA G + + C: Color of colonies F:Fluorescence of colonies G: Growth of colonies L: Localization of stainon colonies eS: External stimulus b-gal: beta-D-galactosidase NA: notapplicable −: absent +/−: weak +: strong ++: very strong

TABLE IVb IO staining of colonies of beta-D-galactosidasepositive/negative bacteria Bacterial colonies after 24 h on NutrientAgar containing 150 mg/l beta-D-galactosidase indicator and 100 mg/lIPTG I16a I21a I22a I25a eS C F C F C F C F Species Strain b-gal y g G yy-g G o o G y o G Aeromonas hydrophila 292 + + + + + + + +/− +/− + ++/− + Citrobacter freundii 181 + + + + + + + + +/− + + + + Enterobacteraerogenes 242 + + + + + + + + +/− + + + + Enterobacter cloacae245 + + + + + + + + +/− + + ++ + Escherichia coli (O157:H7)60 + + + + + + + + +/− + ++ ++ + Escherichia coli 61 + + + + + + + ++/− + ++ ++ + Escherichia coli 270 + + + + ++ + + + +/− + ++ ++ +Klebsiella pneumoniae 164 + + + + + + + + +/− + + + + Serratiamarcescens 40 + + + + ++ + + + +/− + + +/− + Staphylococcus intermedius31 + +/− +/− +/− +/− +/− +/− − − − +/− +/− + Staphylococcussaprophyticus 273 + + + + + + + − − − +/− +/− + Salmonella bongori 81 ++/− +/− + +/− +/− + +/− − + +/− +/− + Salmonella illa 73 + − − + − − + −− + − − + Shigella dysenteriae Serovar 1 131 +/− − − + − − + − − + − − +Shigella sonnei 133 +/− + + + + + + + +/− + + ++ + Yersiniaenterocolitica 175 +/− − − + − +/− + − − + − − + Salmonella enteritidis43 − − − + − − + − − + − − + Bacillus subtilis 65 − − − + − − +/− − − −− − + Listeria monocytogenes (4b) 290 − − − +/− − − +/− − − − − − +/−Pseudomonas aeruginosa 168 − − +¹ + − +¹ + − +¹ + − +¹ + Shigella boydii135 − − − + − − + − − + − − + Staphylococcus aureus 2 − − − +/− − − +/−− − − +/−² − + Streptococcus agalactiae 195 − − − +/− − − +/− − − − − −+/− I4a I28a I27a I31a (Control) eS C F C F C F C F Species Strain b-galo o G y o G r r G b − G Aeromonas hydrophila 292 + +/− − + + + + + − + +− + Citrobacter freundii 181 + + +/− + + + + + +/− + + − + Enterobacteraerogenes 242 + + +/− + + + + + +/− + ++ − + Enterobacter cloacae245 + + +/− + ++ ++ + + +/− + ++ − + Escherichia coli (O157:H7) 60 + ++/− + ++ ++ + + +/− + ++ − + Escherichia coli 61 + + +/− + ++ ++ + ++/− + ++ − + Escherichia coli 270 + ++ +/− + ++ ++ + + +/− + ++ − +Klebsiella pneumoniae 164 + + +/− + + + + + +/− + ++ − + Serratiamarcescens 40 + +/− − + + + + + − + + − + Staphylococcus intermedius31 + + +/− + − − +/− + − + + − + Staphylococcus saprophyticus 273 + +/−− + +/− +/− + + − + + − + Salmonella bongori 81 + +/− − + +/− +/− + +/−− + +/− − + Salmonella illa 73 + − − + − − + − − + − − + Shigelladysenteriae Serovar 1 131 +/− − − + − − + − − + − − + Shigella sonnei133 +/− + +/− + + ++ + + +/− + + − + Yersinia enterocolitica 175 +/− −− + +/− − + +/− − + − − + Salmonella enteritidis 43 − − − + − − + − − +− − + Bacillus subtilis 65 − − − +/− − − + − − + − − + Listeriamonocytogenes (4b) 290 − − − +/− − − +/− − − +/− − − +/− Pseudomonasaeruginosa 168 − − +¹ + − +¹ + − − + − +¹ + Shigella boydii 135 − − − +− − + − − + − − + Staphylococcus aureus 2 − − − +/− − − +/− − − + y² − +Streptococcus agalactiae 195 − − − +/− − − +/− − − +/− − − +/− C: Colorof colonies F: Fluorescence of colonies G: Growth of colonies y: yellowg: green o: orange r: red b: blue p: pink −: absent +/−: weak/variable+: strong ++: very strong ¹blue autofluorescence ² S. aureus naturalcolor

TABLE IVc IO staining of colonies of bacteria producing variousbiomarker enzymes Bacterial colonies after 24 h on Nutrient Agarcontaining 0.45 mM beta-D-Glucuronidase Indicator I3f I21f (control) eSC F C Species Strain glucuronidase y y-o G p F G Escherichia coli 61 + ++/− + + − + Escherichia coli 62 + + +/− + + − + Escherichia coli 270 + ++/− + + − + Klebsiella 164 − − − + − − + pneumoniae Bacterial coloniesafter 24 h on Nutrient Agar containing 150 mg/l alpha-D-GlucosidaseIndicator I21e eS C F Species Strain a-glucosidase y y-g G Enterobactersakazakii 141 + + +/− + Enterobacter sakazakii 142 + + +/− +Enterobacter aerogenes 242 − − − + Enterobacter cloacae 245 − − − +Shigella sonnei 133 − − − + Escherichia coli 270 − − − + Bacterialcolonies after 24 h on Nutrient Agar containing 0.52 mM C1-EsteraseIndicator I4c I19c (control) eS C F C F Species Strain c1-esterase y y Gb − G Klebsiella pneumoniae 164 + * + + − + Escherichia coli 270 +/− * +− − +/− Bacillus cereus 289 ++ * + − − − Bacterial colonies after 24 hon Tryptic Soy Agar containing 0.78 mM PC PLC Indicator, 3.2 g/l BSA,0.05% Tween80 and 5 mM MnCl₂ I4d I25d (control) eS C F C F SpeciesStrain plc y y-g G b − G Bacillus cereus 51 + + ++ + +/− − + Bacilluscereus 289 + + ++ + − − + Bacillus cereus 191 + + ++ + +/− − + Bacilluscereus 192 + ++ ++ + ++ − + Bacillus mycoides 64 + + ++ + + − + Bacillusthuringiensis 66 + + ++ + +/− − + Escherichia coli 61 +/− +/− +/− + +− + Escherichia coli 270 +/− +/− +/− + +/− − + Listeria monocytogenes290 + − − + − − + (4b) Pseudomonas aeruginosa 168 + y-g¹ b² + y-g¹ b² +*: diffuse background due to some precipitation of substrate ¹naturalcolor, same on agar without substrate ²natural fluorescence, same onagar without substrate I21f:1-(2-Benzoylphenyl)-6-chloro-1H-indol-3-yl-beta-D-glucuronide sodiumsalt I3f: 6-Chloro-1H-indol-3-yl-beta-D-glucuronide sodium salt I21e:1-(2-Benzoylphenyl)-6-chloro-1H-indol-3-yl-alpha-D-glucopyranoside I19c:1-(2-Benzoylphenyl)-1H-indol-3-yl acetate I4c:5-Bromo-4-chloro-1H-indol-3-yl acetate I25d:1-[2-(2,4-Dimethoxybenzoyl)phenyl]-1H-indol-3-yl choline phosphate I4d:5-Bromo-4-chloro-1H-indol-3-yl choline phosphate eS: External Stimulusglucuronidase: beta-D-glucuronidase a-glucosidase: alpha-D-glucosidaseplc: phosphatidyl choline phospholipase c C: Color of colonies F:Fluorescence of colonies G: Growth of colonies y: yellow g: green o:orange r: red b: blue p: pink −: absent +/−: weak/variable +: strong ++:very strong

TABLE IVd IN and IO concurrent staining of bacterial colonies NutrientAgar containing beta-D-galactosidase IO indicator I21a and IN indicatorI4h Species Strain eS: b-gal eS: b-glu C G Escherichia coli 270 + − y +Klebsiella pneumoniae 164 + + g + Enterobacter aerogenes 242 + + g +Enterococcus faecalis 210 − + b + I21a:1-(2-Benzoylphenyl)-6-chloro-1H-indol-3-yl-beta-D-galactopyranoside I4h:5-Bromo-4-chloro-1H-indol-3-yl-beta-D-glucopyranoside IO:10H-indolo[1,2-a]indole staining IN: Indigo staining eS: ExternalStimulus b-gal: beta-D-galactosidase b-glu: beta-D-glucosidase −: absent+/−: weak/variable +: strong y: yellow g: green b: blue

TABLE IVe IN and IO staining of bacterial colonies under anaerobicconditions Nutrient Agar plates containing various beta-D-galactosidaseindicators anaerobically incubated with E. coli Indicator Type/System CI L D S G I21a IO y + + +/− 0.7 mm + I22a IO o-r + ++ − 0.5 mm + I27a IOy-o + + +/−   1 mm + I31a IO o-r + +/− + 0.8 mm + I4a  IN b +/− − ++pinpoint +/− IN: Indigo staining IO: 10H-indolo[1,2-a]indole staining C:Color of colonies I: Intensity of color L: Localization on colonies D:Diffusion around colonies S: Size of colonies G: Growth of colonies −:absent +/−: weak/variable +: strong ++: very strong I21a:1-(2-Benzoylphenyl)-6-chloro-1H-indol-3-yl-beta-D-galactopyranosideI22a:1-(2-Benzoylphenyl)-5-bromo-4-chloro-1H-indol-3-yl-beta-D-galactopyranosideI27a:6-Chloro-1-[2-(2,4-dimethoxybenzoyl)phenyl)-1H-indol-3-yl-beta-D-galactopyranosideI31a:6-Chloro-1-[2-(N-methylpyrrole-2-carbonyl)phenyl]-1H-indol-3-yl-beta-D-galactopyranosideI4a: 5-Bromo-4-chloro-1H-indol-3-yl-beta-D-galactopyranoside y: yellowr: red b: blue o: orange

TABLE IVf IO staining of bacterial colonies on Blood Agar platesKlebsiella pneumoniae colonies after 24 h on Blood Agar containing 150mg/l I21a and 100 mg/l IPTG Color of Indicator colonies Color ofcolonies Growth white/visible UV (366 nm) I21a Yellow Yellow-orange on adark background + — Gray Very weak bluish in front of dark + background(hardly visible) IO: 10H-indolo[1,2-a]indole I21a:1-(2-Benzoylphenyl)-6-chloro-1H-indol-3-yl-beta-D-galactopyranoside −:absent +: strong

TABLE IVg IO staining of fungal colonies Agar plates containingindicator I21g eS: Species Strain galactosamidase Color Growth Candidaalbicans 304 + Yellow +/− Candida krusei 305 − Cream +/− Saccharomycescerevisiae 306 − Cream +/− IO: 10H-indolo[1,2-a]indole I21g:1-(2-Benzoylphenyl)-6-chloro-1H-indol-3-yl-N-acetyl-beta-D-galactosaminide−: absent +/−: weak/variable +: strong

The invention claimed is:
 1. An indicator system for detecting anexternal stimulus, comprising an indicator compound of the generalformula

wherein: X is O, NH or S; LG is a labile group with the X-LG moietybeing susceptible to conversion by action of said external stimulus; R₁,R₂, R₃ and R₄ are independently selected from the group consisting ofhydrogen, C1-4 alkyl; C1-4 alkoxy; fused or linearly connected aryl;fused or linearly connected heteroaryl; halogen; cyano; nitro; formyl;and optionally substituted amino, carboxy, carbonyl, hydroxy andsulfonyl; R₅ is either hydrogen or R₁₂, wherein R₁₂ is

wherein: Z is O, NH or S; R₆, R₇, R₈, R₉ and R₁₀ are independentlyselected from the group consisting of hydrogen, C1-4 alkyl; C1-4 alkoxy;fused or linearly connected aryl; fused or linearly connectedheteroaryl; halogen; cyano; nitro; formyl; and optionally substitutedamino, carboxy, carbonyl, hydroxy and sulfonyl; and wherein, if R₅ ishydrogen, the indicator system further comprises an acceptor compound ofthe general formula

wherein: R_(a) and R_(b) are independently selected from hydrogen andC1-4 alkyl.
 2. The indicator system according to claim 1, wherein R₅ ishydrogen and the acceptor compound (B) is2-methoxy-4-(N,N-dimethylamino)benzaldehyde.
 3. The indicator systemaccording to claim 1, wherein LG is selected from the group consistingof beta-D-galactopyranoside, tert-butyldimethylsilyloxy (TBDMS),acetate, choline phosphate, alpha-D-glucopyranoside, beta-D-glucuronidesodium salt, N-acetyl-beta-D-galactosaminide and beta-D-glucopyranoside.
 4. The indicator system according to claim 1, wherein R₁₀ isselected from the group consisting of hydrogen, methyl, methoxy, phenyl,DMP, CFur, Fur, NPyr, wherein:


5. A method of detecting an external stimulus in a region of interest,comprising: providing the region of interest with an indicator system;and monitoring for a signal from a signalophore species formed as aconsequence of said external stimulus; wherein said indicator system isas defined in claim 1 with the X-LG moiety being susceptible toconversion by action of said external stimulus, said conversion leadingto formation of a signalogen species comprising an enolic moiety whereinXH is bound to a carbon atom that is bound to a further carbon atom by adouble bond; said signalophore species being formed by reaction of saidenolic moiety with an acceptor moiety selected from carbonyl, imino andthiocarbonyl.
 6. The method according to claim 5, wherein said acceptormoiety is a carbonyl moiety.
 7. The method according to claim 6, whereinsaid carbonyl moiety is provided by adding an acceptor compound of thegeneral formula

wherein: R_(a) and R_(b) are independently selected from hydrogen andC1-4 alkyl.
 8. The method according to claim 7, wherein the acceptorcompound (B) is 2-methoxy-4-(N,N-dimethylamino)benzaldehyde.
 9. Themethod according to claim 5, wherein said acceptor moiety is a part ofsaid indicator molecule.
 10. The method according to claim 5, wherein,if R₅ is hydrogen, the signalophore species is a 2-benzylideneindolinewith the structural formula

and wherein, if R₅ is R₁₂, the signalophore species is a10H-indolo[1,2-a]indole with the structural formula

wherein R₁₃ is either OH or R₁₀.
 11. The method according to claim 5,carried out under substantially oxygen-depleted conditions.
 12. A methodof preparing an indicator compound of the general formula

wherein: X is O, NH or S; LG is a labile group with the X-LG moietybeing susceptible to conversion by action of said external stimulus; R₁,R₂, R₃ and R₄ are independently selected from the group consisting ofhydrogen, C1-4 alkyl; C1-4 alkoxy; fused or linearly connected aryl;fused or linearly connected heteroaryl; halogen; cyano; nitro; formyl;and optionally substituted amino, carboxy, carbonyl, hydroxy andsulfonyl; and R₅ is R₁₂ which is

wherein: Z is O, NH or S; and wherein R₆, R₇, R₈, R₉ and R₁₀ areindependently selected from the group consisting of hydrogen, C1-4alkyl; C1-4 alkoxy; fused or linearly connected aryl; fused or linearlyconnected heteroaryl; halogen; cyano; nitro; formyl; and optionallysubstituted amino, carboxy, carbonyl, hydroxy and sulfonyl;characterized in that it comprises the step of N-arylation of an indoxylcompound of the general formula

with a benzene derivative of the general formula

wherein Q is a leaving group selected from iodo, bromo, triflate andtosylate.
 13. A compound of structural formula

wherein X is O, NH or S R₁, R₂, R₃, R₄, R₆, R₇, R₈, R₉ and R₁₃ areindependently selected from the group consisting of hydrogen, C1-4alkyl; C1-4 alkoxy; fused or linearly connected aryl; fused or linearlyconnected heteroaryl; halogen; cyano; nitro; formyl; and optionallysubstituted amino, carboxy, carbonyl, hydroxy and sulfonyl, with theexception of the following compounds


14. A method of administering the compound according to claim 13 in anindicator system for detecting an external stimulus, comprisingadministering the compound to a region of interest.
 15. The method ofclaim 12, wherein Q is a leaving group selected from iodo or bromo.